CN103501593A - Cucurbita plant resistant to potyvirus - Google Patents

Cucurbita plant resistant to potyvirus Download PDF

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CN103501593A
CN103501593A CN201280018999.1A CN201280018999A CN103501593A CN 103501593 A CN103501593 A CN 103501593A CN 201280018999 A CN201280018999 A CN 201280018999A CN 103501593 A CN103501593 A CN 103501593A
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potyvirus
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CN103501593B (en
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M·尼克拉斯
J-L·尼科莱特
M·奥利弗
S·达南
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Syngenta Participations AG
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Abstract

The present invention relates to a Cucurbita plant, in particular a squash plant, having wide spectrum resistance to potyvirus such as Zucchini Yellow Mosaic Virus (ZYMV), Watermelon Mosaic Virus (WMV), Papaya Ringspot Virus (PRSV) and Moroccan watermelon mosaic virus (MWMV). Methods of selecting a squash plant having wide spectrum potyvirus resistance by marker assisted breeding are also provided.

Description

The cucurbita plants that Potyvirus is there is to resistance
The present invention relates to that Potyvirus is had to the novel plant of resistance and the seed of described plant.The invention still further relates to and manufactured these plants and for generation of their method of seed.The invention further relates to mark and their purposes in marker-assisted breeding.
Potyvirus group (its prototype member is called marmor upsilon (PVY)) is (Ward and Shu Kela, 1991 of maximum in current 34 plant viruses group of having identified and family; International virology 32,269-296(Ward& Shukla, 1991; Intervirology32,269-296)).This group comprises at least 180 definite and possible members (account for all known plant viruses 30%), and these members cause heavy losses (Ward and Shu Kela, 1991 in agricultural, livestock breeding, gardening and ornamental crops; International virology 32,269-296).
A subject matter in the pumpkin cultivation is the Potyvirus that infringement plant and fruit have occurred.At least four kinds of Potyviruses that the most often infect pumpkin are arranged, little ZYMV (ZYMV), watermelon mosaic virus (WMV), PRSV (PRSV), Morocco's watermelon mosaic virus (MWMV).The symptom of the Potyvirus disease in pumpkin comprises mosaic disease, yellow, elongated shape leaf, hypoevolutism and fruit and seed deformity.
Stable Potyvirus resistance is pumpkin breeder's crucial driving factors, because multiple virus is tended to sudden change and defeated existing resistant gene.In pumpkin, hitherto known unique stable resistance realizes by the genetic modification method.In Europe, the pumpkin planting person sees that in these " resistance " kinds Potyvirus infects day by day.Thereby, prevent need to not being satisfied of strategy that the pumpkin plant is infected by Potyvirus to convenient and continuable economically.
The present invention by provide different Potyvirus on affecting generally pumpkin have stable and widely the pumpkin plant of resistance solve this needs.This resistance is to provide by by classical interspecific cross and embryo, saving at least 3 recessive inheritance determinants that are incorporated in the pumpkin plant.
Summary of the invention
The present invention relates to a kind of cucurbita plants of cultivation, comprising at least one can instruct or control Potyvirus, preferably to one or more the genetic determinant of resistance in MWMV, PRSV, WMV and ZYMV.
In one embodiment, described genetic determinant is from China squash (Cucurbita moschata), preferably obtainable from China squash Nigeria mutation (C.moschata var.Nigeria).
In another embodiment, according to the plant of any previous embodiment comprise described at least one can instruct or control the genetic determinant of the resistance that Potyvirus is infected.
In another embodiment, comprise at least two genetic determinants that can instruct or control the resistance that Potyvirus is infected according to the plant of any previous embodiment.
In another embodiment, comprise at least three according to the plant of any previous embodiment and can instruct or control Potyvirus is infected, the genetic determinant of the resistance preferably ZYMV and MWMV infected.
In another embodiment, the present invention relates to a kind of plant according to any previous embodiment, wherein
Existing corresponding genetic determinant complementation at least one in these genetic determinants and field pumpkin cultivation kind (C.pepo cv.) 268NiW, the representative seed of this field pumpkin cultivation kind 268NiW is kept at NCIMB with accession number NCIMB41727, described corresponding genetic determinant and marker site ZN genetic linkage, this marker site and Potyvirus resistance trait, preferably with ZYMV and PRSV resistance trait be divided into from, and can be by with following Oligonucleolide primers, amplification of DNA fragments being identified it in PCR: forward primer SEQ ID NO:1(5'AGGTTTCATGGGCTTTTAATGG3') and reverse primer SEQ ID NO:2(5'CGTGAGCCTAAAACGGTTAATG3'), FAM-CACTTCCCAGCCCAAAT-MGB-NFQ(SEQ ID NO:7) and/or susceptible allele-specific probe use subsequently the resistance allele specific probe:: VIC-CACTTTCCAGCCCAAAT-MGB-NFQ(SEQ ID NO:8) detected, and/or
In these genetic determinants at least two with field pumpkin cultivation kind 268NiW in existing corresponding genetic determinant complementation, the representative seed of this field pumpkin cultivation kind 268NiW is kept at NCIMB with accession number NCIMB41727, described corresponding genetic determinant and marker site W2 genetic linkage, this marker site and Potyvirus resistance trait, preferably with a kind of MWMV resistance trait be divided into from, and can be by with following Oligonucleolide primers, amplification of DNA fragments being identified it in PCR: forward primer SEQ ID NO:3(5'GGGCAAAGAAGATCTTGTCTAGAAAG3') and reverse primer SEQ ID NO:4(5'GTTTTTGTGCAGTGTGCATCTGT3'), FAM-TCATTGCACCCAACATG-MGB-NFQ(SEQ ID NO:9) and/or susceptible allele-specific probe use subsequently the resistance allele specific probe:: VIC-TCATTGCACTCAACATGG-MGB-NFQ(SEQ ID NO:10) detected,
And/or forward primer SEQ ID NO:5(5'TTGTGTTTATATGTATGTGTGCGAG3') and reverse primer SEQ ID NO:6(5'TTTCTAGATCTCAGTGTAAGAGAACACA3'), FAM-TTTGTTTGCTTGAGCTGG-MGB-NFQ(SEQ ID NO:11) and/or susceptible allele-specific probe use subsequently the resistance allele specific probe:: VIC-TTTGTTCGATTGAGCTGG-MGB-NFQ(SEQ ID NO:12) detected; And/or
Existing corresponding genetic determinant complementation at least one in these genetic determinants and field pumpkin cultivation kind 268NiW, the representative seed of this field pumpkin cultivation kind 268NiW is kept at NCIMB with accession number NCIMB41727, described corresponding genetic determinant and marker site Ni+ genetic linkage, this marker site and Potyvirus resistance trait, preferably with a kind of ZYMV resistance trait, more preferably with a kind of ZYMV Strain Nivir resistance trait be divided into from, and can be by with following Oligonucleolide primers, amplification of DNA fragments being identified it in PCR: forward primer SEQ ID NO:13(5'TTGCATGTTCCTTGGATGGGT3') and reverse primer SEQ ID NO:14(5'GGCAACCTCTGTCCAATTTCTTTC3'), FAM-AGTTGCGACTTTCCA-MGB-NFQ(SEQ ID NO:15) and/or susceptible allele-specific probe use subsequently the resistance allele specific probe:: TET-AGTTGCGACTTTTCATT-MGB-NFQ(SEQ ID NO:16) detected.
In another embodiment, the present invention relates to a kind of plant according to any previous embodiment, wherein
Existing corresponding genetic determinant complementation in this genetic determinant and field pumpkin cultivation kind 268NiW, the representative seed of this field pumpkin cultivation kind 268NiW is kept at NCIMB with accession number NCIMB41727, described corresponding genetic determinant and marker site ZN genetic linkage, this marker site and Potyvirus resistance trait, preferably with a kind of ZYMV and PRSV resistance trait be divided into from, and can be in PCR by with following Oligonucleolide primers to amplification of DNA fragments in field pumpkin cultivation kind 268NiW genome by its evaluation: forward primer SEQ ID NO:1 and reverse primer SEQ ID NO:2, with SEQ ID NO:7 and/or SEQ ID NO:8, detected subsequently,
And/or existing corresponding genetic determinant complementation in these genetic determinants and field pumpkin cultivation kind 268NiW, the representative seed of this field pumpkin cultivation kind 268NiW is kept at NCIMB with accession number NCIMB41727, described corresponding genetic determinant and marker site W1 and W2 genetic linkage, this marker site and Potyvirus resistance trait, preferably with a kind of MWMV resistance trait be divided into from, and can be in PCR by with following Oligonucleolide primers to amplification of DNA fragments in field pumpkin cultivation kind 268NiW genome by its evaluation: forward primer SEQ ID NO:3 and reverse primer SEQ ID NO:4, with SEQ ID NO:9 and/or SEQ ID NO:10, detected subsequently, and/or forward primer SEQ ID NO:5 and reverse primer SEQ ID NO:6, with SEQ ID NO:11 and/or SEQ ID NO:12, detected subsequently, and/or
Existing corresponding genetic determinant complementation in this genetic determinant and field pumpkin cultivation kind 268NiW, the representative seed of this field pumpkin cultivation kind 268NiW is kept at NCIMB with accession number NCIMB41727, described corresponding genetic determinant and marker site Ni+ genetic linkage, this marker site and Potyvirus resistance trait, preferably with a kind of ZYMV resistance trait, more preferably with a kind of ZYMV Strain Nivir resistance trait be divided into from, and can be in PCR by with following Oligonucleolide primers to amplification of DNA fragments in field pumpkin cultivation kind 268NiW genome by its evaluation: forward primer SEQ ID NO:13 and reverse primer SEQ ID NO:14, with SEQ ID NO:15 and/or SEQ ID NO:16, detected subsequently.
In another embodiment, the present invention relates to a kind of plant according to any previous embodiment, wherein
This genetic determinant and marker site ZN genetic linkage, this marker site and Potyvirus resistance trait, preferably with a kind of ZYMV and PRSV resistance trait be divided into from, and can be in PCR by with following Oligonucleolide primers to amplification of DNA fragments in described Plant Genome by its evaluation: forward primer SEQ ID NO:1 and reverse primer SEQ ID NO:2, with SEQ ID NO:7 and/or SEQ ID NO:8, detected subsequently; And/or
This genetic determinant and marker site W1 and/or W2 genetic linkage, this marker site and Potyvirus resistance trait, preferably with a kind of MWMV resistance trait be divided into from, and can be in PCR by with following Oligonucleolide primers to amplification of DNA fragments in described Plant Genome by its evaluation: forward primer SEQ ID NO:3 and reverse primer SEQ ID NO:4, with SEQ ID NO:9 and/or SEQ ID NO:10, detected subsequently;
And/or forward primer SEQ ID NO:5 and reverse primer SEQ ID NO:6, with SEQ ID NO:11 and/or SEQ ID NO:12, detected subsequently; And/or
This genetic determinant and marker site Ni+ genetic linkage, this marker site and Potyvirus resistance trait, preferably with a kind of ZYMV resistance trait, more preferably with a kind of ZYMV Strain Nivir resistance trait be divided into from, and can be in PCR by with following Oligonucleolide primers to amplification of DNA fragments in described Plant Genome by its evaluation: forward primer SEQ ID NO:13 and reverse primer SEQ ID NO:14, with SEQ ID NO:15 and/or SEQ ID NO:16, detected subsequently.
In another embodiment, the present invention relates to a kind of plant according to any previous embodiment, wherein should (these) genetic determinant be recessive inheritance and separate independently of one another.
In another embodiment, the present invention relates to a kind of plant according to any previous embodiment, wherein this plant is the pumpkin plant.
The invention still further relates to the seed of a kind of plant of the pumpkin according to any previous embodiment, this seed can grow into Potyvirus resistance pumpkin plant.The invention still further relates to the purposes of described seed, for growing into Potyvirus resistance pumpkin plant.
The invention still further relates to a kind ofly for generation of to Potyvirus, preferably at least one in MWMV, PRSV, WMV and ZYMV represented to the method for the pumpkin plant of resistance, comprise the following steps:
A) select a kind ofly to comprise at least one and can instruct or control the pumpkin plant to the genetic determinant of the resistance of Potyvirus, wherein said genetic determinant be obtainable from the genome of field pumpkin cultivation kind 268NiW and with at least one marker site genetic linkage, this at least one marker site with the resistance of Potyvirus is divided into from, and can be in PCR with at least one following PCR Oligonucleolide primers to by its evaluation:
I. forward primer SEQ ID NO:1 and reverse primer SEQ ID NO:2, detected with SEQ ID NO:7 and/or SEQ ID NO:8 subsequently; And/or
Ii. forward primer SEQ ID NO:3 and reverse primer SEQ ID NO:4, detected with SEQ ID NO:9 and/or SEQ ID NO:10 subsequently; And/or
Iii. forward primer SEQ ID NO:5 and reverse primer SEQ ID NO:6, detected with SEQ ID NO:11 and/or SEQ ID NO:12 subsequently; And/or
Iv. forward primer SEQ ID NO:13 and reverse primer SEQ ID NO:14, detected with SEQ ID NO:15 and/or SEQ ID NO:16 subsequently; And/or
V. a forward and reverse primer, it can identify with resistance to Potyvirus be divided into from a marker site;
B) make step described plant and easy infection Potyvirus a) or at least one described Potyvirus represented to the pumpkin plant hybridization of medium resistance level; And
C) select an offspring from described hybridization, this offspring represents a kind of resistant phenotype to Potyvirus, and wherein said resistant phenotype separates with step described at least one marker site a).
The invention still further relates to a kind of for generation of according to any previous embodiment Potyvirus is represented to the method for the pumpkin plant of resistance, comprise the following steps:
A) select a kind ofly to comprise at least one and can instruct or control Potyvirus, preferably to the pumpkin plant of the genetic determinant of the resistance one of at least in ZYMV and MWMV, wherein said genetic determinant be obtainable from the genome of field pumpkin cultivation kind 268NiW and with at least one marker site genetic linkage, this at least one marker site and Potyvirus resistance be divided into from, and can be in PCR with comprising that at least one following PCR Oligonucleolide primers is to by its evaluation:
If i. this marker site is ZN, be forward primer SEQ ID NO:1 and reverse primer SEQ ID NO:2, with SEQ ID NO:7 and/or SEQ ID NO:8, detected subsequently; Or
Ii. forward primer SEQ ID NO:3 and reverse primer SEQ ID NO:4, detected with SEQ ID NO:9 and/or SEQ ID NO:10 subsequently; And/or
If iii. this marker site is W2, be forward primer SEQ ID NO:5 and reverse primer SEQ ID NO:6, with SEQ ID NO:11 and/or SEQ ID NO:12, detected subsequently; And/or
If iv. this marker site is Ni+, be forward primer SEQ ID NO:13 and reverse primer SEQ ID NO:14, with SEQ ID NO:15 and/or SEQ ID NO:16, detected subsequently;
B) make step described plant and easy infection Potyvirus a) or at least one described Potyvirus represented to the pumpkin plant hybridization of medium resistance level; And
C) select an offspring from described hybridization, this offspring represents a kind of resistant phenotype to Potyvirus, and wherein said resistant phenotype separates with step described at least one marker site a).
In one embodiment, the present invention relates to a kind of method according to any previous embodiment, this plant of wherein selecting in a) in step comprises at least two and can instruct or control the genetic determinant to the resistance of Potyvirus; And at step c) in this offspring of selecting to Potyvirus, preferably ZYMV and MWMV are represented to a kind of resistant phenotype, and wherein said resistant phenotype is with two marker sites a) separate in step.
In another embodiment, the present invention relates to a kind of method according to any previous embodiment, the plant of wherein selecting in a) in step comprises at least three can be instructed or control Potyvirus, preferably to the genetic determinant of the resistance of ZYMV and MWMV; And at step c) in this offspring of selecting to Potyvirus, preferably ZYMV and MWMV are represented to a kind of resistant phenotype, and wherein said resistant phenotype separates with step three marker sites a).
In another embodiment, the present invention relates to a kind of method according to any previous embodiment, wherein step donor plant a) is a Plants as described in this.
In another embodiment, the present invention relates to a kind of method according to any previous embodiment, comprise and make step c) in this virus resistance plant and the step b that obtain) this susceptible pumpkin plant or the additional step that backcrosses of medium resistance pumpkin plant.
The invention still further relates to a kind of method of hybrid seed of the pumpkin for generation of Potyvirus being there is to resistance, comprise a kind of male sterile female of plantation and a kind ofly malely can educate plant, at least one in wherein said male or female strain is plant as described in this, thereby realize cross pollination between two strains, make this plant growth until setting is collected these fruits and obtained these hybrid seeds.
The invention still further relates to a kind ofly for obtaining the method for Potyvirus resistance pumpkin plant, comprise
A) by a kind of method as described in this, obtain as described a Plants at this;
B) make plant or a kind of medium resistance pumpkin plant hybridization of described plant and a kind of easy infection Potyvirus;
C) save by step b) an embryo producing of this hybridization;
D) from step c) described embryo bear again a kind of plant; And
E) select Potyvirus is had the steps d of resistance) a Plants.
The invention still further relates to and a kind ofly for obtaining, Potyvirus is had to the method for the pumpkin fruit of resistance, comprise sowing as described in this or the seed of the Plants by a kind of acquisition of method as described in this; With make described plant growth in order to produce fruit and this fruit that results are produced by described plant.
The invention still further relates to and a kind ofly can instruct or control the genetic determinant to the resistance of Potyvirus, wherein said genetic determinant is obtainable from the genome of field pumpkin cultivation kind 268NiW, and wherein
Existing corresponding genetic determinant complementation in this genetic determinant and field pumpkin cultivation kind 268NiW, the representative seed of this field pumpkin cultivation kind 268NiW is kept at NCIMB with accession number NCIMB41727, described corresponding genetic determinant and marker site ZN genetic linkage, this marker site and Potyvirus resistance trait, preferably with a kind of ZYMV and PRSV resistance trait be divided into from, and can be by with following Oligonucleolide primers, amplification of DNA fragments being identified it in PCR: forward primer SEQ ID NO:1 and reverse primer SEQ ID NO:2, with SEQ ID NO:7 and/or SEQ ID NO:8, detected subsequently, and/or
Existing corresponding genetic determinant complementation in this genetic determinant and field pumpkin cultivation kind 268NiW, the representative seed of this field pumpkin cultivation kind 268NiW is kept at NCIMB with accession number NCIMB41727, described corresponding genetic determinant and marker site W2 genetic linkage, this marker site and Potyvirus resistance trait, preferably with a kind of MWMV resistance trait be divided into from, and can be by with following Oligonucleolide primers, amplification of DNA fragments being identified it in PCR: forward primer SEQ ID NO:3 and reverse primer SEQ ID NO:4, with SEQ ID NO:9 and/or SEQ ID NO:10, detected subsequently,
And/or forward primer SEQ ID NO:5 and reverse primer SEQ ID NO:6, with SEQ ID NO:11 and/or SEQ ID NO:12, detected subsequently; And/or
Existing corresponding genetic determinant complementation in this genetic determinant and field pumpkin cultivation kind 268NiW, the representative seed of this field pumpkin cultivation kind 268NiW is kept at NCIMB with accession number NCIMB41727, described corresponding genetic determinant and marker site Ni+ genetic linkage, this marker site and Potyvirus resistance trait, preferably with a kind of ZYMV resistance trait, more preferably with a kind of ZYMV Strain Nivir resistance trait be divided into from, and can be by with following Oligonucleolide primers, amplification of DNA fragments being identified it in PCR: forward primer SEQ ID NO:13 and reverse primer SEQ ID NO:14, with SEQ ID NO:15 and/or SEQ ID NO:16, detected subsequently.
The invention still further relates to a kind of evaluation comprises at least one and can instruct or control Potyvirus, preferably to the method for the pumpkin plant of the genetic determinant of the resistance one of at least in ZYMV and MWMV, wherein said genetic determinant be obtainable from the genome of field pumpkin cultivation kind 268NiW and with at least one marker site genetic linkage, at least one in this at least one marker site and described Potyvirus resistant phenotype be divided into from, and can be in PCR by use, comprise that following PCR Oligonucleolide primers is to by its evaluation:
If this marker site is ZN, be forward primer SEQ ID NO:1 and reverse primer SEQ ID NO:2, with SEQ ID NO:7 and/or SEQ ID NO:8, detected subsequently; Or
If this marker site is W1, be forward primer SEQ ID NO:3 and reverse primer SEQ ID NO:4, with SEQ ID NO:9 and/or SEQ ID NO:10, detected subsequently; And/or
If this marker site is W2, be forward primer SEQ ID NO:5 and reverse primer SEQ ID NO:6, with SEQ ID NO:11 and/or SEQ ID NO:12, detected subsequently; Or
If this marker site is Ni+, be forward primer SEQ ID NO:13 and reverse primer SEQ ID NO:14, with SEQ ID NO:15 and/or SEQ ID NO:16, detected subsequently.
The invention still further relates to the purposes of the Potyvirus resistance propagating materials that derives from a kind of plant of pumpkin as described in this, for growing into a kind of Potyvirus resistance pumpkin plant in order to produce fruit and gather in the crops described fruit.
Definition
As used herein, phrase " breeding population of foundation " refers at breeding plan, the set for example produced in the business breeding plan and/or be used as parent's potential breeding gametophyte.The member of the breeding population of setting up is typically fully being characterized aspect gene and/or phenotype.For example, may assess interested some phenotypic characters, for example under different environmental conditions, in a plurality of positions and/or in the different time.As an alternative or in addition, may identify the relevant gene loci of expression one or more and phenotypic character, and may be should one or more gene locis aspect and just the one or more genetic markers aspect relevant to these one or more gene locis one or more members of breeding population are carried out to genotyping.
It is no longer to be native state that the custard squash plant of cultivation is understood to mean within the scope of the invention, but by the mankind's treatment, has been grown and tamed and supply agricultural use and/or human consumption's plant.As an example, custard squash plant according to the present invention will be considered to a kind of plant of cultivation, and can be selected from lower group, this group comprises jewel pumpkin, zucchini, butternut squash, little custard squash, yellow bent neck pumpkin, yellow zucchini, custard squash, fan-shaped pumpkin, straight neck pumpkin and snake melon.In the context of the present invention, " the custard squash plant of cultivation " is suitable for cultivation and represents disease resistance, particularly medium resistance, as little ZYMV (ZYMV) medium resistance.The cucurbita plants of cultivation should further be understood to not comprise the wild type species of those proterties that comprised theme of the present invention as natural character and/or its natural genetic part.
For fear of doubt, the mutation of China squash Nigeria is resistance " wild source " and the plant that should not be considered to a kind of cultivation.
As used herein, phrase " dliploid individuality " refers to the individuality with two group chromosomes, and typically, one group from each in its two parents.Yet, should understand in certain embodiments, the dliploid individuality can receive from identical single creature body the chromosome of its " female parent " and " male parent " group, as when the autologous pollination of plant during with the subculture of generation plant.
" isozygoty " and be interpreted as within the scope of the invention referring to the identical allelomorph of the one or more corresponding site on homologous chromosome.
" heterozygosis " is interpreted as referring to the different allelomorph of the one or more corresponding site on homologous chromosome within the scope of the invention.
" backcrossing ", to be interpreted as within the scope of the invention be the method for instigating one of hybrid generation and parent to repeat back to hybridize.Different recurrent parents can be used in backcrossing subsequently.
" site " is interpreted as within the scope of the invention referring on chromosome and comprises gene or to a zone of contributive any other gene element of proterties or key element.
As used herein, " marker site " refers on chromosome to have comprised and is present in individual genome and a zone of the nucleotide relevant to one or more interested sites or polynucleotide sequence, and this zone may comprise gene or to contributive any other gene element of proterties or key element." marker site " also refers on chromosome to comprise the zone with the polynucleotide sequence (as the sequence of the nucleic acid as probe) of genome sequence complementation.
" genetic linkage " is interpreted as referring to the contiguous hereditary feature relevance caused in due to the gene position on same chromosome within the scope of the invention, by the restructuring percentage between site recently measure (centimorgan, cM).
Distance between site is measured by the exchange frequency between the site on same chromosome usually.It is far away that two sites are separated by, and between them, just more may exchange.Otherwise, if two sites closely are close together, between them, unlikely exchange.By convention, a centimorgan (cM) equals the restructuring of 1% between site (mark).When QTL can indicate by a plurality of marks, the size of the Gene distance indication QTL between the end points mark.
Phrase " with marker site genetic linkage " will be considered to mean marker site and provide the genetic determinant of resistance trait to be no more than 10cM apart, more preferably 5cM, more preferably 2cM, most preferably 1cM.
" instruct or control the genetic determinant of expressing " is interpreted as referring at this can be on the level of DNA self, the expression to proterties on the translation of final polypeptide product, the level transcribing and/or activate has contribution, thereby causes heritable gene element of the phenotypic expression of this proterties.
For purposes of the present invention, term " be divided into from " refers to the following fact: for the allelomorph of proterties with transmit together with allelomorph for mark tends to, this is because they physically abut against together (restructuring between them is because the vicinity of their physics reduces) on same chromosome, causes their allelomorph contiguous and nonrandom association on same chromosome due to them." be divided into from " also refers in single plant exist two or more proterties, known wherein at least one be heredity and these proterties can not by contingency, explain easily.
As used herein, term " gene structure of qualitative character site " refers to relevant with interested phenotypic character on statistics and is representing the genome area of the hereditary basis of interested phenotypic character.
As used herein, refer in the context of the theme that phrase " sexual hybridization " and " sexual reproduction " disclose in the present invention that Gamete Fusion is to produce offspring's (for example,, by fertilization, as in plant, by pollination, produced seed)." sexual hybridization " or " cross fertilization " be in certain embodiments one by one body for example, by another individual fertilization (, the cross pollination in plant).Term " self-fertilization " refers in certain embodiments by self-fertilization or autologous pollination and produces seed; That is, pollen and ovule are from same plant.
As used herein, phrase " genetic marker " refers to feature relevant to one or more interested sites in individual genome (for example, being present in nucleotide or the polynucleotide sequence in individual genome).In certain embodiments, genetic marker is polymorphism in interested colony, or site occupied by polymorphism, depends on context.Genetic marker comprises that for example single nucleotide polymorphisms (SNP), insertion or disappearance (that is, insertion/deletion), simple sequence repeat (SSR), restrictive fragment length polymerphism (RFLP), randomly amplified polymorphic DNA (RAPD), cracking amplification polymorphism sequence (CAPS) mark, diversity array technique (DArT) mark and amplified fragment length polymorphism (AFLP) and many other examples.Genetic marker can be for example for to comprising the gene loci on chromosome, the contributive allelomorph of the changeability of phenotypic character positioned.Phrase " genetic marker " can also refer to the polynucleotide sequence with the genome sequence complementation, as the sequence of the nucleic acid as probe.
Phrase " with existing corresponding gene group determinant complementation in field pumpkin cultivation kind 268NiW " will be considered to mean a gene (or its promoter region) of finding on a zone of chromosomal DNA, the length in described zone is 0.5MB, 1MB, 2MB, 3MB, 4MB, 5MB or 10MB, and this gene is consistent with the homologous genes (or its promoter region) of finding on respective regions at field pumpkin cultivation kind 268NiW chromosomal DNA.
Genetic marker can physically be positioned a position of inner at the gene loci associated with it on chromosome or outside (that is being, intragenic or extragenic accordingly).In other words, for example, although the position on chromosome (at the gene corresponding to interested site or function mutation, in the control element of gene outside) typically use a plurality of genetic markers while also there is no between identified and genetic marker and interested site the non-zero recombination fraction, but the theme that the present invention discloses can also be used genetic marker in the border in gene loci physically (for example, in the genome sequence inside corresponding to gene, as but be not limited to the intron of gene or the polymorphism in exon).In some embodiment of the theme disclosed in the present invention, these one or more genetic markers are included in the mark between and ten, and in certain embodiments, these one or more genetic markers comprise more than ten genetic markers.
As used herein, term " genotype " is the genomic constitution of phalangeal cell or organism.Individual " genotype of one group of genetic marker " comprises the concrete allelomorph in the one or more genetic markers site existed in individual haplotype.As known in the art, genotype can relate to single site or a plurality of site, and no matter these sites are relevant or uncorrelated, and/or is that the chain right and wrong of going back are chain.In certain embodiments, individual genotype relates to one or more relevant genes, the expression of (for example, quantity or qualitative character) as defined herein because the interested phenotype of one or more participations in these genes.Thereby in certain embodiments, genotype comprises one or more allelic the gathering that one or more gene locis place of individual inherent quantity or qualitative character exists.In certain embodiments, genotype is expressed from haplotype (being defined in hereinafter) aspect.
As used herein, term " germplasm " refers to the genotypic overall of a colony or other group of individuals (for example, species).Term " germplasm " can also refer to vegetable material; For example, one group of plant that serves as different allelic storages.Phrase " germplasm of transformation " for example refers to for given environment or area to have the vegetable material through the hereditary superiority of proof, and phrase " the not germplasm of transformation ", " original germplasm " and " Exotic Germplasm " refer to for example for given environment or area, have vegetable material unknown or that uncertified heredity is worth; Thereby phrase " the not germplasm of transformation " refers to the part that is not the breeding population set up and the vegetable material that does not have known relation with the member of the breeding population of setting up in certain embodiments.
As used herein, term " hybrid ", " hybrid plant " and " hybrid generation " refer to the individuality that produced by the parent different in heredity (for example, in heredity heterozygosis or be mainly the individuality of heterozygosis).
As used herein, phrase " single hybridization F1 hybrid " refers to the F1 hybrid produced by the hybridization between two inbred line.
As used herein, phrase " inbred line " refers to the colony of isozygotying that isozygoty in heredity or intimate.For example, the brother that inbred line can be by some circulations/sister's breeding or self-fertilization or double haploid produce to obtain.In certain embodiments, inbred line carries out pure breeding for one or more interested phenotypic characters." inbreeding ", " inbreeding individuality " or " inbreeding offspring " are the independent samples from an inbred line.
As used herein, term " double haploid strain " refers to the stable inbred line drawn by the flower pesticide cultivation.Some pollen grains (monoploid) of cultivating in special media and environment can develop into and contain n chromosomal plantlet.Then make these plantlets " double " and comprise 2n chromosome.The offspring of these plantlets is called " double haploid " and basically no longer separates (stable).
As used herein, term " chain " and its grammatical variants refer to the allelomorph of site different on same chromosome in the situation that their transmission is independent tending to separates more frequently than accidentally desired.
As used herein, phrase " nucleic acid " refers to may be corresponding to any physics monomeric unit string of nucleotide string, (for example comprise the polymer of nucleotide, typical DNA, cDNA or RNA polymer), the oligonucleotides of modified (for example, comprise the oligonucleotides for biological RNA or the atypical base of DNA, as the 2'-O-oligonucleotides that methylates) etc.In certain embodiments, nucleic acid can be sub-thread, bifilar, multiply or their combination.Unless otherwise instructed, otherwise the complementary series that the concrete nucleotide sequence of the theme that the present invention discloses optionally comprises or encode except any sequence of clearly indicating.
As used herein, phrase " phenotypic character " refers in individuality outward appearance or other the detectable features that the interaction by individual genome, protein group and/or metabolism group and environment produces.
As used herein, phrase " resistance " refers to that when with the susceptible plants comparison plant can be limited advolution and/or their caused damages of specifying pathogene under similarly environmental condition and pathogen pressure.Resistance plant, at pathogen pressure, for example may represent some diseases symptom or damage under the ZYMV pathogen pressure.
As used herein, phrase " neurological susceptibility " refers to that plant can not limit the appointment pathogene fully, and Potyvirus pathogene for example, as the advolution of ZYMV.
To show nothing or few downright bad and nothing or extremely rare sporulation under the test condition that resistance plant defines in following instance.
As used herein, term " a plurality of " refers to more than one.Thereby " a plurality of individuality " refers at least two individualities.In certain embodiments, the term majority refers to half more than integral body.For example, in certain embodiments, " majority in colony " refers to half more than the member of that colony.
As used herein, term " offspring " refers to one or more filial generations of concrete hybridization.Typically, the offspring is produced by the breeding of two individualities, but some species (particularly some plants and hermaphroditic animal) can self-fertilization (that is, same plant serves as male and donor female gamete).The one or more offspring can be for example F1, F2 or any subculture.
As used herein, phrase " qualitative character " refers to the phenotypic character of the gene control that is subject to one or several to represent most of phenotype effects.Therefore, qualitative character is typically by heredity simply.Example in plant includes but not limited to the color of flower, color and some known disease resistances of fruit.
" selection based on mark " is interpreted as within the scope of the invention referring to and for example uses genetic marker to detect one or more nucleic acid from plant, wherein this nucleic acid and desirable character inheritance are chain, to identify the plant of the gene that carries (or undesirable) proterties of making us hope, make in selection breeding and can use in the works (or avoiding) those plants.
" polymerase chain reaction (PCR) " is interpreted as referring to the relatively a large amount of concrete zone that produces genomic DNA or one or more subsets within the scope of the invention, thus the method for carrying out the possible different analysis based on those zones.
" PCR primer " is interpreted as referring to the relatively short single-stranded DNA fragment of using in the pcr amplification in concrete zone of DNA within the scope of the invention.
" phenotype " is interpreted as referring to the differentiable feature of the upper proterties of controlling of heredity within the scope of the invention.
As used herein, phrase " phenotypic character " refers in individuality outward appearance or other the detectable features that the interaction by individual genome, protein group and/or metabolism group and environment produces.
" polymorphism " is interpreted as within the scope of the invention referring in a colony and has two or more multi-form genes, genetic marker or genetic character or such as by obtainable gene outcomes such as alternately montage, DNA methylations.
" selection breeding " is interpreted as referring to using to have or the breeding plan of the plant of the proterties of wishing as the parent made us in demonstration within the scope of the invention.
" test " plant is interpreted as referring to for characterizing the plant of the proterties of tested plant in heredity within the scope of the invention.Typically, make tested plant and " test " plant hybridization, and the segregation ratio of the proterties in filial generation is marked.
" probe " refers to and can identify and be incorporated into objectives molecule or cell structure and thereby allow one group of atom or the molecule of the detection of target molecule or structure as used in this.Especially, " probe " refer to can be for detecting the existence of complementary series and quantitative DNA or RNA sequence through mark to it by molecular hyridization.
Term " hybridization " refers to conventional hybridization conditions as used in this, preferably refers to following hybridization conditions, that is: use 5x SSPE, 1%SDS, 1x Denhardts solution as solution and/or hybridization temperature between 35 ℃ and 70 ℃, preferably 65 ℃.After hybridization, preferably at first with 2x SSC, 1%SDS and subsequently with 0.2x SSC at the temperature between 35 ℃ to 75 ℃, especially between 45 ℃ to 65 ℃ but especially under 59 ℃, washed (about the definition of SSPE, SSC and Denhardts solution, referring to the people such as Pehanorm Brooker (Sambrook) in above-mentioned quoted passage).As the high stringency hybridization conditions described in the people such as above Pehanorm Brooker is particularly preferred.Particularly preferred stringent hybridization condition is for example being hybridized as above instructions and washing existence while carrying out under 65 ℃.For example hybridize and to wash the low stringency hybridization condition of carrying out under 45 ℃ be not too preferred and under 35 ℃, be not too preferred.
" sequence homology or sequence identity " used interchangeably at this.Under the background of two or more nucleic acid or protein sequence, term " unanimously " or " uniformity " percentage refer to as used one of following sequence comparison algorithm or being measured by range estimation, when comparing and comparing maximum correspondence, two or more sequences or subsequence are identical or have identical amino acid residue or the nucleotide of prescribed percentage.If two sequence length differences that will compare each other, sequence identity preferably relates to the percentage of the nucleotide residue of the shorter sequence consistent with the nucleotide residue of longer sequence.Routinely by use computer program for example the Bestfit program (Wisconsin sequence analysis bag, for the version 8 of Unix operating system, genetics computer group, the university research garden, No. 575, scientific precursor road, Madison, WI53711) can determine sequence identity.Bestfit is used Smith and Whatman, applied mathematics progress 2(1981), 482-489(Smith and Waterman, Advances in Applied Mathematics2 (1981), local homology's algorithm 482-489) has the conforming section of maximal sequence in order to find between two sequences.When determining whether that by Bestfit or other sequence alignment programs a concrete sequence and a canonical sequence of the present invention for example have 95% uniformity, thus preferably to parameter carry out such adjustment on the whole length of canonical sequence, conforming percentage is calculated and allow this canonical sequence in the autoploidy breach account for the nucleotide sum up to 5%.When using Bestfit, preferably so-called optional parameter is retained in to their default (" acquiescence ") values.Between the sequence of a given sequence and the invention described above relatively in the deviation that occurs may cause by for example adding, lack, replace, insert or recombinating.Preferably can also service routine " fasta20u66 " (version 2 .0u66, in September, 1998, by William R. Pearson came (William R.Pearson) and University of Virginia, write, also referring to W.R. Pearson came (1990), Enzymology method 183,63-9a(W.R.Pearson (1990), Methods in Enzymology183,63-9a), appended example and http://workbench.sdsc.edu/) carry out a kind of like this sequence relatively.For this purpose, can use " default " setting parameter.Two another consistent in fact indications of nucleotide sequence are the hybridization each other under stringent condition of these two kinds of molecules.Phrase " specific hybrid " refers to that a molecule only is combined with a specific nucleotide sequence under stringent condition, two strandsization or hybridization, and this is for example, to carry out in this sequence is present in a kind of compound mixture (, total cell) DNA or RNA the time." in fact in conjunction with " refer in a probe nucleic acid and the complementation between a target nucleic acid and hybridize, and contain a small amount of mispairing, and these mispairing can be held by the stringency that reduces this hybridization medium, to realize the desirable detection of this target nucleic acid sequence.
Nucleic acid hybridization experiment (as, south and north hybridization) background under " stringent hybridization condition " and " strictly hybridizing wash conditions " be sequence-dependent, and be different under different environmental parameters.Longer sequence is specifically hybridized at higher temperature.The extensive guidance of nucleic acid hybridization is seen to hybridization the 2nd chapter part i " Hybridization principle and inspection of nucleic acid probes strategy summary " of gloomy (1993) biochemistry of the base of a fruit and Molecular Biology Lab's technology-use nucleic acid probe, like to think only that, New York (Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes part I chapter2 " Overview of principles of hybridization and the strategy of nucleic acid probe assays " Elsevier, New York).In general, for the ion strength limiting and the concrete sequence under the pH value, high strict hybridization and wash conditions are selected as lower approximately 5 ℃ than heat fusion joint.Typically, under " stringent condition ", probe will be hybridized with its target subsequences, but not can with other sequence hybridizations.
Temperature (under the ion strength limited and pH value) when heat fusion joint is 50% target sequence with the Probe Hybridization of coupling fully.Utmost point stringent condition is selected as equaling the T for concrete probe m.The example for the stringent hybridization condition of the hybridization that has the complementary nucleic acid that surpasses 100 complementary residues in south or northern blot assay on filter is under 42 ℃, and 50% formamide containing the 1mg heparin, wherein spent the night hybridization.An example of highly strict wash conditions is 0.15M NaCl, under 72 ℃, continues approximately 15 minutes.An example of strict wash conditions is under 65 ℃, 0.2x SSC washing, continue 15 minutes (about the explanation of SSC buffer solution, referring to the Pehanorm Brooker, below).Often, can first hang down the stringency washing before high stringency washing, to remove the background probe signals.An example that the duplex that for example surpasses 100 nucleotide is carried out to medium stringency washing is under 45 ℃, and 1x SSC continues 15 minutes.An example that the duplex that for example surpasses 100 nucleotide is hanged down to the stringency washing is under 40 ℃, and 4-6x SSC continues 15 minutes.For short probe (for example, about 10 to 50 nucleotide), stringent condition typically relates to the salinity of the Na ion that is less than about 1.0M, typically approximately 0.01 to the Na ion concentration (or other salts) of 1.0M, at pH7.0 to 8.3 time, and this temperature is typically at least about 30 ℃.Can also realize stringent condition by adding stable reagent (as formamide).In general, in concrete hybridization check, signal to noise ratio is that 2 times (or higher) indication observed for uncorrelated probe detects specific hybrid.If the coded albumen of the nucleic acid of not hybridizing each other under stringent condition is consistent in fact, they remain consistent in fact.For example, while, when the maximum codon degeneracy that uses genetic code to allow, generating a nucleic acid copy, this thing happens.
" plant " is any plant, particularly spermatophyte in any stage of growing.
" plant cell " is structure and the physiology unit of plant, comprises protoplast and cell wall.Plant cell can be the form of single cell or the cultured cell of separation, or as the part of high systematism unit (as plant tissue, plant organ or whole strain plant).
" plant cell cultures " means the culture of plant unit (as for example protoplast, the cell in cell culture cell, the plant tissue in the different developmental stage, pollen, pollen tube, ovule, blastular, joint element and embryo).
" vegetable material " or " from the obtainable vegetable material of plant " refers to any other part or the product of part, fruit, pollen, egg cell, joint element, seed, slitting, cell or tissue culture or the plant of leaf, stem, root, flower or flower.
" plant organ " is uniqueness of plant and the obvious part of structuring and differentiation, as root, stem, leaf, bud or embryo.
" plant tissue " means one group of plant cell that tissue changes into the 26S Proteasome Structure and Function unit as used herein.Comprise any plant tissue in plant or in culture.This term includes but not limited to: whole strain plant, plant organ, plant seed, tissue culture and being organized changes into any plant cell group of structure and/or functional unit.In conjunction with as plant tissue or the other plant tissue of being contained by this definition of any particular type listed above or do not have the plant tissue that uses at that time this term not plan to get rid of any other type.
Detailed description of the invention
The present invention relates to novel pumpkin plant, these pumpkin plants infect and to have resistance Potyvirus, and thereby avoid suffering the infringement by due to this pathogene.The invention still further relates to and manufacture and use the method for these plants.
According to plant of the present invention, can obtain by hybridizing two or more parent genotypes, at least one in these genotype can have one or more allelomorph, particularly to one or more allelomorph of the contributive corresponding site of Potyvirus resistance, this one or more allelomorph lacks other parent genotypes or supplements other genotype to obtain according to the present invention and plant as described in this.If the expression more than a site antagonism proterties has contribution and two original parent genotypes that whole group of allelomorph is not provided, in breeding population, can comprise other sources.Other parent genotypes can contribution make us the proterties of wishing, comprise the required crop quality in market.
Parent genotype can hybridize to produce progeny seed each other.Parent genotype can be that the uncontrolled or open pollination of the heterozygosis plant utilization by selecting from field is carried out self-fertilization and used recurrent selection procedure to grow the inbred line formed.Make outstanding plant self-fertilization and selected in subculture.In these subcultures, as the result of autologous pollination and selection, the heterozygosis condition provides approach for the homogeneity strain.Utilize the subculture of inbreeding, plant becomes and more and more isozygotys and homogeneous in progeny plants.Typically, (F1 is to F2 can to put into practice five to seven generations that self-fertilization and pedigree select or more generations; F3 is to F4; F4 is to F5) with obtain Plants and Seeds feature homogeneous and continue under self-fertilization will homogeneous inbred line.
In the process of inbreeding, many undesirable allelomorph of heterozygosis site will be by more favourable allelic replacement, and eliminates disadvantageous or undesirable allelomorph from the offspring.In addition, by the selection based on mark, can make favourable allelic number maximize, wherein identify more disadvantageous allelomorph and in succession use more favourable allelic replacement.
In one aspect, can be by making can instruct or control the genetic determinant of the resistance of Potyvirus is penetrated in the pumpkin plant (the particularly custard squash plant of cultivation) of cultivation and obtains from ancestors plant (particularly wild ancestors plant) according to plant of the present invention.
In a specific embodiment of the present invention, can therefrom obtain the wild ancestors that can instruct or control the genetic determinant of the resistance of Potyvirus is the wild Chinese pumpkin, particularly wild Chinese pumpkin Nigeria mutation, or its offspring or the ancestors that carry out self-contained described genetic determinant.In replacement scheme, according to Potyvirus resistance trait of the present invention, (this proterties provides Potyvirus is infected to the plant of expressing this proterties, preferably to one or more the resistance in MWMV, PRSV, WMV and ZYMV) can be kept at NCIMB with accession number NCIMB41727 from its representative seed of field pumpkin cultivation kind 268NiW(), or obtain from offspring or the ancestors that can instruct or control the genetic determinant of the resistance of Potyvirus comprising of field pumpkin cultivation kind 268NiW.
Therefore, in a specific embodiment of the present invention, to the contributive parent genotype of Potyvirus resistance trait, it is a kind of inbred line, it has the correlation properties of the field pumpkin cultivation kind 268NiW that the present invention preserves,, the genome identical in fact in the site be associated with the Potyvirus resistance constructed, and the seed sample of field pumpkin cultivation kind 268NiW is kept at NCIMB on June 14th, 2010 with accession number NCIMB41727.Field pumpkin cultivation kind 268NiW has resistance to MWMV, PRSV, WMV and ZYMV.Resistance is to be provided by genetic determinant Zn, Ni+, W1 and W2.These determinants recessive inheritance of isozygotying in 268NiW.These determinants are transferable between a plurality of genetic backgrounds.Resistance assay proves, for example, in these genetic determinants background (yellow pumpkin) different at these, continues to provide the resistance of wide spectrum for Potyvirus.Resistance level to different potyvirus strains is shown in Table 2.When with Potyvirus, attacking plant, the beneficial effect with all 4 genetic determinants is shown in table 2 and Figure 10.
Upper to the contributive potential of hybrid generation for practicality and its heredity of determining inbred line, test hybridization with another inbred line, and the gained offspring is assessed on phenotype.
In another specific embodiment of the present invention, the contributive parent genotype of antagonism proterties is a kind of hybrid, it has the correlation properties of the field pumpkin cultivation kind 268NiW that the present invention preserves,, the genome identical in fact in the site be associated with the Potyvirus resistance constructed, and the seed sample of field pumpkin cultivation kind 268NiW is kept at NCIMB on June 14th, 2010 with accession number NCIMB41727.
Field pumpkin cultivation kind 268NiW is that donor and the custard squash inbred line cross as the Potyvirus resistance trait produces by the mutation of wild Chinese pumpkin Nigeria.By the Potyvirus resistance offspring of hybridization specifically and other inbred line crosses of different genes background finally to obtain field pumpkin cultivation kind 268NiW.
Therefore, field pumpkin cultivation kind 268NiW or comprise any other plant strain that can instruct or control the genetic determinant of the resistance of Potyvirus and can be used as source material, to obtain according to the present invention, Potyvirus is infected to the pumpkin plant with highly resistant for making described resistance trait penetrate into any desirable genetic background, may further include the one or more proterties of wishing, crop quality proterties as required as market of making us.Except crop quality, also has important feature on agronomy, as for example good plant structure, high yield and to the basic resistance of pathogene.
Based on description of the invention, have its sample of field pumpkin cultivation kind 268NiW(as described in this and with accession number NCIMB41727, be kept at NCIMB Co., Ltd) or comprise at least one and can instruct or control and be not difficult to use breeding technique well known in the art described at least one genetic determinant of the present invention to be transferred to other pumpkin plants of different types to its offspring of the genetic determinant of the resistance of Potyvirus or ancestors' technical staff.Proterties of the present invention can for example be transferred to other Cucurbitas.Therefore, in one embodiment, plant of the present invention is to belong to the pumpkin plant infected by resistant to PVY.In one embodiment of the invention, make the pumpkin plant growth to obtain (hybrid) seed or to produce for the business pumpkin.
Therefore, in another embodiment, the present invention disclose a kind of will according to of the present invention at least one can instruct or control the method for the genetic determinant of the resistance of Potyvirus being transferred to the pumpkin plant that lacks described proterties, comprise and a) obtain the plant that comprises described proterties; B) make it and the plant hybridization that lacks described proterties; The plant of hybridization c) acquisition step b); D) the plant selective basis step c infected that can resistant to PVY belongs to of the present invention).In one embodiment, the method further comprises e) make by steps d) plant and the pumpkin plant that produce backcross, and f) the selective basis pumpkin plant that can resistant to PVY belongs to infection of the present invention.In one embodiment, the method further comprises obtaining and a kind ofly according to the present invention, can belong to the inbreeding pumpkin plant infected by resistant to PVY, and in one embodiment, the method further comprises and makes described inbreeding pumpkin plant and another pumpkin plant hybridization a kind ofly according to of the present invention, can belong to the hybrid pumpkin plant infected by resistant to PVY to produce.In one embodiment, as in this description, the pumpkin plant is exist or do not exist Potyvirus to select by determining.In one embodiment, step plant a) that comprises described proterties is that its representative seed of field pumpkin cultivation kind 268NiW(is kept at NCIMB with accession number NCIMS41727) or offspring or the ancestors of described plant.
Can also the usage flag assistant breeding identify and comprise at least one and can instruct or control those individualities to the genetic determinant of the resistance of Potyvirus and/or side joint marker site as the described herein or the marker site chain with it.
Selection based on mark may be used to the commitment that inbreeding is grown, and often with the screening technique based on can visually determining and relate to plant phenotypic characteristic of relevant Key Performance Indicator for the adaptability of business hybrid production to a great extent, combines.Selection can also be based on molecular labeling, these molecular labelings may with or may be not and the interested linkage of characters.
Especially, the selection based on mark can or be applied those individualities that all have favourable homozygous genotype with all related locus of the present invention described here before identifying wherein with the Phenotypic Selection combination after Phenotypic Selection.
There is the molecular labeling of some types can be used to the selection based on mark, include but not limited to the random amplification (RAPD) of restrictive fragment length polymerphism (RFLP), polymorphic dna and the restrictive fragment length polymerphism (AFLP) of amplification.
RFLP relates at concrete short restriction site and cuts chromosomal DNA with restriction enzyme, by between these positions copy or lack or the sudden change at these restriction site places produces polymorphism.
RAPD utilizes low stringency polymerase chain reaction (PCR) to increase and produces the Strain specificity array of anonymous DNA fragment with the single primer with arbitrary sequence.The method only needs DNA sample seldom and analyzes a large amount of pleomorphism sites.
AFLP need to use restriction enzyme vitellophag DNA before the concrete fragment of selectivity amplification oligonucleotide in using PCR and primer.The technology of the fragment of utilizing this method, range estimation to obtain, can measure nearly 100 pleomorphism sites for each combination of primers, and each test only needs a small amount of DNA sample.
Ssr analysis is based on microsatellite DNA (short weight the is multiple) sequence extensively be distributed in Eukaryotic genome, the variation during these sequence selective ground amplifications are repeated with the detection simple sequence.Ssr analysis only needs DNA sample seldom.SNP is used the PCR that can effectively obtain point mutation to extend check.The every duplicate samples of this program needs few DNA.One or both can use said method in the typical selection breeding plan based on mark in.
The most preferred method of amplification that has realized crossing over the nucleotide fragments in plant genome polymorphism district is used polymerase chain reaction (" PCR ") (people such as Mu Lisi, cold spring port quantitative biology discussion 51:263-273(1986) (Mullis et al., Cold Spring Harbor Symp.Quant.BioI.51:263273 (1986))), it has used the paired primer that comprises a forward primer and a reverse primer, and these primers can be its bifilar form and the contiguous sequence hybridization that has defined polymorphism.
Can carry out amplified fragments by alternative, as " ligase chain reaction " (" LCR ") (Ba Lani, the periodical 88:189-193(1991 of institute of NAS) (Barany, Proc.Natl.Acad.Sci. (U.SA) 88:189193 (1991)), it carrys out index amplification objectives with two pairs of oligonucleotide probes.The sequence of every a pair of oligonucleotides is through selecting to allow same one the contiguous sequence hybridization of this pair of and this target.This hybridization has formed the substrate of template dependence ligase.As PCR, so products therefrom serves as template in circulation subsequently, and obtains the index amplification of desirable sequence.
Can utilize have the polymorphism position same one near-end and the oligonucleotides of far-end sequence carry out LCR.In one embodiment, arbitrary oligonucleotides will be designed to include the actual polymorphism position of polymorphism.In such an embodiment, select reaction condition so that oligonucleotides only in the situation that target molecule comprises or shortage and oligonucleotides on the specificity nucleotide of the polymorphism position complementation that exists can be joined together.As an alternative, oligonucleotides can be selected so that they do not comprise polymorphism position (referring to match lattice (Segev), PCT applies for WO90101069).
Can be used as other method that replacement scheme used and be " oligonucleotides connects check " (" OLA ") people such as (, science 241:1077-1080(1988) Lan Degelun (Landegren et aI., Science241:10771080 (1988))).The OLA scheme has been used two oligonucleotides that are designed to the contiguous sequence hybridization of the sub-thread of target.As LCR, OLA is particularly suitable for the test point sudden change.Yet, being different from LCR, OLA obtains " linearity " of target sequence and non-exponential amplification.
The other method again that can be used as the replacement scheme use is " intrusive mood check ", the three-dimensional composite that its uses structure specificity sheet endonuclease (FEN) to come cracking to be formed with the target dna hybridization that comprises single nucleotide polymorphisms (SNP) position by the overlapping oligonucleotides of allele-specific.Make with target molecule in the oligonucleotides annealing of SNP allelic complementation trigger the cracking of oligonucleotides by heat endurance FEN cracking.Cracking can detect by some diverse ways.Modal, pyrolysis product triggers second pyrolysis and reacts to discharge fluorescence signal on FRET (fluorescence resonance energy transfer) (FRET) box.As an alternative, cracking can be by being used fluorescence polarization (FP) probe or directly detecting by mass spectral analysis.Cracking reaction has high degree of specificity, has low mortality, and can detect the target dna of 10-21 mole.Although this check is being used to the SNP of every secondary response in inquiring about a duplicate samples traditionally, but, through test, novel has made it become a kind of check effectively and accurately that is suitable for multichannel high flux SNP genotyping based on chip or the approach based on bead.
The people such as Nickerson (Nickerson) have described the check of a kind of detection of nucleic acids, and it has combined attribute people such as (, the periodical 87:8923-8927(1990 of institute of NAS) Nickersons of PCR and OLA).In this method, PCR is used to the index amplification of realize target DNA, then uses OLA to detect.
Connect two (or more) oligonucleotides under the existence of the nucleic acid of the sequence based on thering is gained " two oligonucleotides ", thereby the scheme of this two oligonucleotides that increases is also known (Wu Dengren, genome 4:560569(1989) (Wu et al., and can be for purpose of the present invention by easily transformation Genomics4:560569 (1989))).
In one embodiment, molecular labeling is the DNA fragmentation by pcr amplification, for example SSR mark or RAPD mark.In one embodiment, through the existence of DNA fragmentation amplification or do not have the specific allelic existence that shows proterties self or this proterties or do not exist.In one embodiment, show the specific allelic existence of proterties through the difference in length of DNA fragmentation of amplification, and thereby make it possible to distinguish the not iso-allele of proterties.
In a specific embodiment of the present invention, the SNP mark is used to identify in mother plant and/or its ancestors and can instructs or control the genetic determinant of the present invention to the resistance of Potyvirus in the progeny plants that the hybridization of described mother plant produces.The SNP mark is detectable by end points reading Taqman technology, and, except specific forward and reverse primer sequence, can also use probe (for example, as disclosed in this) to detect the existence of R and/or the S-allele at each marker site place.So, each in disclosed genetic marker be by four rather than only two sequences form: the amplification forward of target area and reverse primer and two probes identifying target SNP.
In the present invention, with resistance to Potyvirus be divided into from of the present invention one or more DNA markers can in PCR, use by following at least one PCR Oligonucleolide primers formed by its evaluation:
I. forward primer SEQ ID NO:1 and reverse primer SEQ ID NO:2, detected with SEQ ID NO:7 and/or SEQ ID NO:8 subsequently; And/or
Ii. forward primer SEQ ID NO:3 and reverse primer SEQ ID NO:4, detected with SEQ ID NO:9 and/or SEQ ID NO:10 subsequently; And/or
Iii. forward primer SEQ ID NO:5 and reverse primer SEQ ID NO:6, detected with SEQ ID NO:11 and/or SEQ ID NO:12 subsequently; And/or
Iv. forward primer SEQ ID NO:13 and reverse primer SEQ ID NO:14, detected with SEQ ID NO:15 and/or SEQ ID NO:16 subsequently,
Described primer produces a kind of amplified production in a PCR reaction, it represented basically with in the PCR reaction that utilizes consistent paired primer at from the obtainable corresponding pcr amplification product of field pumpkin cultivation kind 268NiW consistent molecular weight or nucleotide sequence, maybe can be regarded as the allelomorph of this corresponding pcr amplification product, the sample of field pumpkin cultivation kind 268NiW is kept at NCIMB Co., Ltd with accession number NCIMB41727.
In a first step, DNA or cDNA sample are from suitable vegetable material, as leaf texture obtains by using known technology extraction DNA or RNA.Then, one of side joint comprises the primer that can instruct or control the zone of the SNP in the genome area of the resistance of Potyvirus disclosed here and is used to use polymerase chain reaction (PCR) method to carry out the DNA amplification sample.
In replacement scheme, desirable allelic existence or do not exist and can determine by the PCR in real time with the distrand DNA dyestuff or fluorescence report sonde method.
Labeled analysis can carry out from the leaf texture of utmost point young plant or from the DNA sample of seed extraction at the development of plants early application.This it is to allow early stage evaluation at breeding cycle to have the plant of the genomic constitution wished and abandoned before pollination not comprise the relevant allelic plant of desirable invention, thereby reduces the size of breeding population and reduce the requirement of phenotype analytical.
In addition, by using molecular labeling, can distinguish isozygoty plant and the heterozygosis plant of only carrying a copy and do not comprise the plant of favourable allelic any copy of instructing or control, at least one genetic determinant place of the resistance of Potyvirus being carried to the relevant allelic two parts of copies of desirable invention.
Thereby, therefore can develop surrogate markers and for the identification of with selective basis the present invention and the allelomorph with qualitative character site as disclosed in this or one group of allelic plant.For example, can obtain the nucleotide sequence of the amplified production obtained by following mode: use by the following PCR Oligonucleolide primers pair formed in pcr amplification:
I. forward primer SEQ ID NO:1 and reverse primer SEQ ID NO:2, detected with SEQ ID NO:7 and/or SEQ ID NO:8 subsequently; And/or
Ii. forward primer SEQ ID NO:3 and reverse primer SEQ ID NO:4, detected with SEQ ID NO:9 and/or SEQ ID NO:10 subsequently; And/or
Iii. forward primer SEQ ID NO:5 and reverse primer SEQ ID NO:6, detected with SEQ ID NO:11 and/or SEQ ID NO:12 subsequently; And/or
Iv. forward primer SEQ ID NO:13 and reverse primer SEQ ID NO:14, detected with SEQ ID NO:15 and/or SEQ ID NO:16 subsequently,
And new definite nucleotide sequence of PCR-based amplified production designs new primer or paired primer.In addition, according to the present invention and before mark disclosed here can be located on the genome of pumpkin or other species, and identical or homology or directly the known mark mapping in the homology zone can be used as developing the starting point of new mark.
Therefore, the mark clearly disclosed in the present invention can also be used to identify and/or develop the mark that the resistance with to Potyvirus new or that add is associated, the recombinant that these are new or then additional mark can be used to again marker-assisted breeding and/or find side joint Potyvirus resistance site, and/or fine Structure Mapping, and/or clone Potyvirus resistance site.
Several available method or way are arranged, they can be used to identify and/or development is linkage disequilibrium and/or with interested region linkage and/or be arranged in the mark in interested zone, and the mark that is representing the actual cause and effect sudden change that causes the Potyvirus resistance trait.In very not comprehensive situation, some approach comprise:
-identify other sequences in interested zone with disclosed sequence/mark in the hybridization approach.
-identify other sequences in interested zone with disclosed sequence/mark in the PCR approach.
-identify other sequences in interested zone with disclosed sequence/mark in the PCR approach.
-mark in mapping and/or relatively mapping approach in identifying same area with disclosed sequence/mark (locate on other collection of illustrative plates described at least one can instruct or control the genetic determinant to the resistance of Potyvirus).
-identify additional sequence/mark/(candidate) gene with disclosed sequence/mark in " being undertaken by calculator " approach.
-disclosed sequence/the mark (at physical mapping or genome sequence, listing the described genetic determinant in location) of use in the physical mapping approach.
-use disclosed sequence/be marked at described at least one genetic determinant in location on other (physics) collection of illustrative plates or genome.
-use the suitable individuality of disclosed sequence/Marker selection, allow to identify the mark in interested zone by the gene approach.
-use disclosed information searching (locational) candidate gene.
For genotyping, mapping or associated mapping, DNA is from suitable vegetable material, as for example extracted in leaf texture.Specifically, collect large quantities of leaves of a plurality of plants.Use multiple polymorphism SSR, SNP or cover genomic any other the suitable type of whole pumpkin the DNA sample is carried out to genotyping.
The Conjoint Analysis of genotype and phenotypic data can be carried out by Application standard software.Can for disclosed here corresponding at least one can instruct or control the allelomorph to the genetic determinant place of the resistance of Potyvirus, mark based on the chain marker site place of described at least one genetic determinant or known nucleotide sequence and these allelic molecular weight that are positioned at any other mark on same chromosome, screen plant introduction and germplasm by one or more technology disclosed here or known to persons of ordinary skill in the art.
Mark, linked marker or disclosed here at least one can instruct or control and can determine by method known to the skilled the nucleotide sequence of the genetic determinant of the resistance of Potyvirus.For example, can be from Potyvirus resistance donor plant, those fragments that genome and the selection by the described plant of fragmentation possesses the mark of described at least one genetic determinant of one or more indications are isolated and are comprised that described at least one genetic determinant or its resistance give the nucleotide sequence of part.Subsequently, or as an alternative, indicate the flag sequence (or its part) in described resistance site can be used as (PCR) amplimer, so that the one or more nucleotide sequences that comprise described resistance site from the amplification of the genomic nucleic acid sample available from described plant or genomic fragment.The nucleotide sequence of described at least one genetic determinant and/or any additional marking of wherein comprising can obtain by the standard sequence measurement.
Therefore, the invention still further relates to a kind of nucleic acid (be preferably DNA but be not limited to DNA) sequence of separation, it comprise of the present invention at least one can instruct or control and give part to genetic determinant or its resistance of the resistance of Potyvirus.Thereby disclosed mark can be used to from pumpkin or other vegetable crops to identify and the mark or the gene that separate one or more or coding Potyvirus resistances chain with the Potyvirus resistance.
With of the present invention described at least one can instruct or control the nucleotide sequence of the additional marking chain to the genetic determinant of the resistance of Potyvirus can also be for example by determining the nucleotide sequence of the one or more marks that are associated with described at least one genetic determinant, and, for the described primers of described mark, then described primer can be used to further determine that the sequence of described mark outside resolves.For example, SNP mark disclosed here or estimate in the zone of described at least one genetic determinant and/or can be checked order by the pcr amplification product to described mark by method well known in the art with the nucleotide sequence of any other mark of described region linkage, or as an alternative in a PCR with described flag sequence or be used as hybridization probe with by screen to identify that chain nucleotide sequence obtains such as but not limited to BAC.
Seed is preserved details
On June 14th, 2010 is kept at following seed sample the NCIMB(Ferguson Building of the Cathy Ferguson mansion, Carlos Kleiber stone villa garden of AB219YA Bake, Britain Scotland Aberdeenshire Si Baien according to the regulation of budapest treaty (the Budapest Treaty) with the name of Syngenta Co.,Ltd (Syngenta Participations AG), Craibstone Estate, Bucksburn, Aberdeen AB219YA, Scotland, UK):
NCIMB41726 field pumpkin cultivation kind PP415
NCIMB41727 field pumpkin cultivation kind 268NiW
Embodiments of the invention
1. the cucurbita plants of a cultivation, custard squash preferably, comprising at least one can instruct or control Potyvirus, preferably to one or more the genetic determinant of resistance in MWMV, PRSV, WMV and ZYMV.
2. according to the plant of embodiment 1, wherein said genetic determinant is from China squash, and preferably the mutation of China squash Nigeria is obtainable.
3. according to the plant of embodiment 1 or 2, wherein said genetic determinant is to exist with a kind of homozygotic state.
4. according to the plant of embodiment 1 to 3, comprise at least one described genetic determinant that can instruct or control the resistance that Potyvirus is infected.
5. according to the plant of embodiment 1 to 3, comprise at least two described genetic determinants that can instruct or control the resistance that Potyvirus is infected.
6. according to the plant of embodiment 1 to 3, comprise at least four described can instruct or control Potyvirus is infected, the genetic determinant of the resistance preferably ZYMV and MWMV infected.
7. according to the plant of any previous embodiment, wherein
A) existing corresponding genetic determinant complementation in this genetic determinant and field pumpkin cultivation kind 268NiW, the representative seed of this field pumpkin cultivation kind 268NiW is kept at NCIMB with accession number NCIMB41727, described corresponding genetic determinant and marker site ZN genetic linkage, this marker site and Potyvirus resistance trait, preferably with a kind of ZYMV and PRSV resistance trait be divided into from, and can be by with following Oligonucleolide primers, amplification of DNA fragments being identified it in PCR: forward primer SEQ ID NO:1 and reverse primer SEQ ID NO:2, with SEQ ID NO:7 and/or SEQ ID NO:8, detected subsequently, and/or
B) two complementations in existing corresponding genetic determinant in this genetic determinant and field pumpkin cultivation kind 268NiW, the representative seed of this field pumpkin cultivation kind 268NiW is kept at NCIMB with accession number NCIMB41727, described corresponding genetic determinant and marker site W1 and/or W2 genetic linkage, these marker sites and Potyvirus resistance trait, preferably with a kind of MWMV resistance trait be divided into from, and can be by with following Oligonucleolide primers, amplification of DNA fragments being identified it in PCR: forward primer SEQ ID NO:3 and reverse primer SEQ ID NO:4, with SEQ ID NO:9 and/or SEQ ID NO:10, detected subsequently,
And/or forward primer SEQ ID NO:5 and reverse primer SEQ ID NO:6, with SEQ ID NO:11 and/or SEQ ID NO:12, detected subsequently; And/or
C) existing corresponding genetic determinant complementation in this genetic determinant and field pumpkin cultivation kind 268NiW, the representative seed of this field pumpkin cultivation kind 268NiW is kept at NCIMB with accession number NCIMB41727, described corresponding genetic determinant and marker site Ni+ genetic linkage, this marker site and Potyvirus resistance trait, preferably with a kind of ZYMV resistance trait, more preferably with a kind of ZYMV Strain Nivir resistance trait be divided into from, and can be by with following Oligonucleolide primers, amplification of DNA fragments being identified it in PCR: forward primer SEQ ID NO:13 and reverse primer SEQ ID NO:14, with SEQ ID NO:15 and/or SEQ ID NO:16, detected subsequently.
8. according to the plant of any previous embodiment, wherein
A) existing corresponding genetic determinant complementation in this genetic determinant and field pumpkin cultivation kind 268NiW, the representative seed of this field pumpkin cultivation kind 268NiW is kept at NCIMB with accession number NCIMB41727, described corresponding genetic determinant and marker site ZN genetic linkage, this marker site and Potyvirus resistance trait, preferably with a kind of ZYMV and PRSV resistance trait be divided into from, and can be in PCR by with following Oligonucleolide primers to amplification of DNA fragments in field pumpkin cultivation kind 268NiW genome by its evaluation: forward primer SEQ ID NO:1 and reverse primer SEQ ID NO:2, with SEQ ID NO:7 and/or SEQ ID NO:8, detected subsequently, and/or
B) two complementations in existing corresponding genetic determinant in this genetic determinant and field pumpkin cultivation kind 268NiW, the representative seed of this field pumpkin cultivation kind 268NiW is kept at NCIMB with accession number NCIMB41727, described corresponding genetic determinant and marker site W1 and/or W2 genetic linkage, these marker sites and Potyvirus resistance trait, preferably with a kind of MWMV resistance trait be divided into from, and can be in PCR by with following Oligonucleolide primers to amplification of DNA fragments in field pumpkin cultivation kind 268NiW genome by its evaluation: forward primer SEQ ID NO:3 and reverse primer SEQ ID NO:4, with SEQ ID NO:9 and/or SEQ ID NO:10, detected subsequently,
And/or forward primer SEQ ID NO:5 and reverse primer SEQ ID NO:6, with SEQ ID NO:11 and/or SEQ ID NO:12, detected subsequently; And/or
C) existing corresponding genetic determinant complementation in this genetic determinant and field pumpkin cultivation kind 268NiW, the representative seed of this field pumpkin cultivation kind 268NiW is kept at NCIMB with accession number NCIMB41727, described corresponding genetic determinant and marker site Ni+ genetic linkage, this marker site and Potyvirus resistance trait, preferably with a kind of ZYMV resistance trait, more preferably with a kind of ZYMV Strain Nivir resistance trait be divided into from, and can be in PCR by with following Oligonucleolide primers to amplification of DNA fragments in field pumpkin cultivation kind 268NiW genome by its evaluation: forward primer SEQ ID NO:13 and reverse primer SEQ ID NO:14, with SEQ ID NO:15 and/or SEQ ID NO:16, detected subsequently.
9. according to the plant of any previous embodiment, wherein
A) this genetic determinant and marker site ZN genetic linkage, this marker site and Potyvirus resistance trait, preferably with a kind of ZYMV and PRSV resistance trait be divided into from, and can be in PCR by with following Oligonucleolide primers to amplification of DNA fragments in described Plant Genome by its evaluation: forward primer SEQ ID NO:1 and reverse primer SEQ ID NO:2, with SEQ ID NO:7 and/or SEQ ID NO:8, detected subsequently; And/or
B) this genetic determinant and marker site W2 genetic linkage, this marker site and Potyvirus resistance trait, preferably with a kind of MWMV resistance trait be divided into from, and can be in PCR by with following Oligonucleolide primers to amplification of DNA fragments in described Plant Genome by its evaluation: forward primer SEQ ID NO:3 and reverse primer SEQ ID NO:4, with SEQ ID NO:9 and/or SEQ ID NO:10, detected subsequently;
And/or forward primer SEQ ID NO:5 and reverse primer SEQ ID NO:6, with SEQ ID NO:11 and/or SEQ ID NO:12, detected subsequently; And/or
C) this genetic determinant and marker site Ni+ genetic linkage, this marker site and Potyvirus resistance trait, preferably with a kind of ZYMV resistance trait, more preferably with a kind of ZYMV Strain Nivir resistance trait be divided into from, and can be in PCR by with following Oligonucleolide primers to amplification of DNA fragments in described Plant Genome by its evaluation: forward primer SEQ ID NO:13 and reverse primer SEQ ID NO:14, with SEQ ID NO:15 and/or SEQ ID NO:16, detected subsequently.
10. according to the plant of any previous embodiment, should (these) genetic determinant be wherein recessive inheritance.
11., according to the plant of any previous embodiment, wherein this plant is a kind of non-transgenic pumpkin plant.
12., according to the plant of any previous embodiment, wherein this plant is an inbred line, a double haploid or a hybrid.
13., according to the plant of any previous embodiment, wherein this plant is male sterile.
A 14. plant material, obtainable from the plant according to any previous embodiment, include but not limited to any other part or the product of part, fruit, pollen, egg cell, joint element, seed, slitting, cell or tissue culture or this plant of leaf, stem, root, flower or flower, this vegetable material still represents a kind of Potyvirus resistant phenotype, particularly when growing into a plant.
15., according to a plurality of plant parts of the plant of any previous embodiment, include but not limited to plant seed, plant organ (as such as root, stem, leaf, bud or embryo etc.), ovule, pollen microspore, plant cell, plant tissue, plant cell cultures (as cell such as in protoplast, cell culture cell, plant tissue, pollen, pollen tube, ovule, blastular, joint element and embryo in the different developmental stage etc.); These plant parts still represent a kind of Potyvirus resistant phenotype, particularly when growing into a plant.
16. the seed of the plant of the pumpkin according to any previous embodiment, this seed can grow into a kind of Potyvirus resistance pumpkin plant.
17., according to the seed of embodiment 16, wherein said seed is hybrid seed.
18., according to the seed of embodiment 17, with accession number 41727, be kept at NCIMB Co., Ltd.
19. the purposes of the seed of an embodiment 16 to 18, for growing into a kind of Potyvirus resistance pumpkin plant.
20. one kind for detection of the kit that can instruct or control in the pumpkin plant the genetic determinant of the resistance of Potyvirus, wherein said kit comprises a PCR Oligonucleolide primers or a PCR Oligonucleolide primers pair that can increase with the chain DNA marker of this genetic determinant, and wherein said DNA marker can be with being selected from a following PCR Oligonucleolide primers to being increased in a PCR:
A) forward primer SEQ ID NO:1 and reverse primer SEQ ID NO:2, detected with SEQ ID NO:7 and/or SEQ ID NO:8 subsequently; And/or
B) forward primer SEQ ID NO:3 and reverse primer SEQ ID NO:4, detected with SEQ ID NO:9 and/or SEQ ID NO:10 subsequently; And/or
C) forward primer SEQ ID NO:5 and reverse primer SEQ ID NO:6, detected with SEQ ID NO:11 and/or SEQ ID NO:12 subsequently; And/or
D) forward primer SEQ ID NO:13 and reverse primer SEQ ID NO:14, detected with SEQ ID NO:15 and/or SEQ ID NO:16 subsequently.
21. a DNA marker, with can instruct or control chain to the genetic determinant of the resistance of Potyvirus and can be with being selected from a following PCR Oligonucleolide primers to being increased in a PCR:
A) forward primer SEQ ID NO:1 and reverse primer SEQ ID NO:2, detected with SEQ ID NO:7 and/or SEQ ID NO:8 subsequently; And/or
B) forward primer SEQ ID NO:3 and reverse primer SEQ ID NO:4, detected with SEQ ID NO:9 and/or SEQ ID NO:10 subsequently; And/or
C) forward primer SEQ ID NO:5 and reverse primer SEQ ID NO:6, detected with SEQ ID NO:11 and/or SEQ ID NO:12 subsequently; And/or
D) forward primer SEQ ID NO:13 and reverse primer SEQ ID NO:14, detected with SEQ ID NO:15 and/or SEQ ID NO:16 subsequently.
22., according to the purposes of any DNA marker of embodiment 21, select for diagnostic that pumpkin is a kind of can instruct or control the genetic determinant to the resistance of Potyvirus.
23. according to the purposes of embodiment 21 to 22 described any DNA markers, for the identification of in plant a kind of can instruct or control to the existence of the genetic determinant of the resistance of Potyvirus and/or for monitoring this gene that can instruct or control the genetic determinant of the resistance of Potyvirus of pumpkin infiltrate.
24. polynucleotides are obtainable with the following methods: in a PCR, by use, be selected from a following PCR Oligonucleolide primers to the DNA fragmentation that increases:
A) forward primer SEQ ID NO:1 and reverse primer SEQ ID NO:2, detected with SEQ ID NO:7 and/or SEQ ID NO:8 subsequently; And/or
B) forward primer SEQ ID NO:3 and reverse primer SEQ ID NO:4, detected with SEQ ID NO:9 and/or SEQ ID NO:10 subsequently; And/or
C) forward primer SEQ ID NO:5 and reverse primer SEQ ID NO:6, detected with SEQ ID NO:11 and/or SEQ ID NO:12 subsequently; And/or
D) forward primer SEQ ID NO:13 and reverse primer SEQ ID NO:14, detected with SEQ ID NO:15 and/or SEQ ID NO:16 subsequently,
Described polynucleotides be included in statistics upper relevant and thereby with a kind of a kind of DNA marker that can instruct or control the genetic determinant genetic linkage of the resistance of Potyvirus, and wherein said polynucleotides are corresponding to utilizing identical paired primer from the obtainable a kind of amplified production of field pumpkin cultivation kind 268NiW in a PCR, the representative seed of this field pumpkin cultivation kind 268NiW is kept at NCIMB with accession number NCIMB41727, its condition is, corresponding marker site still is present in described field pumpkin cultivation kind 268NiW plant, and/or can be regarded as its allelomorph.
25. polynucleotides, have at least 90%, particularly at least 95%, particularly at least 96%, particularly at least 97%, particularly at least 98%, particularly at least 99% sequence identity with the sequence of the polynucleotides of embodiment 24.
26. polynucleotides, represent a kind of nucleotide sequence with the nucleotide sequence hybridization of the polynucleotides of embodiment 25.
27. one kind for instructing at least one or control the method that the genetic determinant of the resistance of Potyvirus is incorporated into to the pumpkin plant that lacks described genetic determinant, comprising:
A) obtain first a pumpkin plant according to any one previous embodiment;
B) make described the first pumpkin plant and second a pumpkin plant hybridization, wherein said the second pumpkin plant lacks described allelomorph; And
C) identify a kind of resistance to Potyvirus that represents increase produced by this hybridization and comprise at least one and described Potyvirus resistance be divided into from the plant of DNA marker; And
D) optionally, separate described plant; And
E) optionally, described plant and this first or second pumpkin plant are backcrossed.
28. one kind for obtaining the method according to the seed of the plant of any previous embodiment, comprises the following steps:
A) obtain first a pumpkin plant according to any one previous embodiment;
B) make described the first pumpkin plant and second a pumpkin plant hybridization, wherein said the second pumpkin plant lacks described genetic determinant; And
C) identify a kind of resistance to Potyvirus that represents increase produced by this hybridization and comprise at least one and described Potyvirus resistance be divided into from the plant of DNA marker; And
D) from described hybridization results comprise at least one and described Potyvirus resistance be divided into from the progeny seed of DNA marker.
29. according to any one method in embodiment 27 or 28, wherein at step c) in, a kind of produce and comprise at least one by this hybridization can instruct or control plant to the genetic determinant of the resistance of Potyvirus and can in PCR, by use, be selected from a following PCR Oligonucleolide primers amplification of DNA fragments is identified it:
A) forward primer SEQ ID NO:1 and reverse primer SEQ ID NO:2, detected with SEQ ID NO:7 and/or SEQ ID NO:8 subsequently; And/or
B) forward primer SEQ ID NO:3 and reverse primer SEQ ID NO:4, detected with SEQ ID NO:9 and/or SEQ ID NO:10 subsequently; And/or
C) forward primer SEQ ID NO:5 and reverse primer SEQ ID NO:6, detected with SEQ ID NO:11 and/or SEQ ID NO:12 subsequently; And/or
D) forward primer SEQ ID NO:13 and reverse primer SEQ ID NO:14, detected with SEQ ID NO:15 and/or SEQ ID NO:16 subsequently.
30., according to the method for embodiment 29, wherein determine the fragment size of the amplified production of one or more paired primers.
31. one kind for generation of to Potyvirus, preferably one or more in MWMV, PRSV, WMV and ZYMV is represented to the method for the pumpkin plant of resistance, comprises the following steps:
A) select a kind ofly to comprise at least one and can instruct or control the pumpkin plant to the genetic determinant of the resistance of Potyvirus, wherein said genetic determinant be obtainable from the genome of field pumpkin cultivation kind 268NiW and with at least one marker site genetic linkage, this at least one marker site with the resistance of Potyvirus is divided into from, and can be in PCR with comprising that at least one following PCR Oligonucleolide primers is to by its evaluation:
I. forward primer SEQ ID NO:1 and reverse primer SEQ ID NO:2, detected with SEQ ID NO:7 and/or SEQ ID NO:8 subsequently; And/or
Ii. forward primer SEQ ID NO:3 and reverse primer SEQ ID NO:4, detected with SEQ ID NO:9 and/or SEQ ID NO:10 subsequently; And/or
Iii. forward primer SEQ ID NO:5 and reverse primer SEQ ID NO:6, detected with SEQ ID NO:11 and/or SEQ ID NO:12 subsequently; And/or
Iv. forward primer SEQ ID NO:13 and reverse primer SEQ ID NO:14, detected with SEQ ID NO:15 and/or SEQ ID NO:16 subsequently; And/or
V. a forward and reverse primer, it can identify with described Potyvirus resistant phenotype at least one be divided into from a marker site;
B) make step described plant and a kind of easy infection Potyvirus a) or at least one described Potyvirus represented to the pumpkin plant hybridization of medium resistance level; And
C) select an offspring from described hybridization, this offspring represents a kind of resistant phenotype to Potyvirus, and wherein said resistant phenotype separates with step described at least one marker site a).
32. according to embodiment 31 for generation of to Potyvirus, preferably, to the method that one of at least represents the pumpkin plant of resistance in ZYMV and MWMV, comprise the following steps:
A) select a kind ofly to comprise at least one and can instruct or control the pumpkin plant to the genetic determinant of the resistance of Potyvirus, wherein said genetic determinant be obtainable from the genome of field pumpkin cultivation kind 268NiW and with at least one marker site genetic linkage, this at least one marker site with the resistance of Potyvirus is divided into from, and can be in PCR with comprising that at least one following PCR Oligonucleolide primers is to by its evaluation:
If i. this marker site is ZN, be forward primer SEQ ID NO:1 and reverse primer SEQ ID NO:2, with SEQ ID NO:7 and/or SEQ ID NO:8, detected subsequently; And/or
If ii. this marker site is W1, be forward primer SEQ ID NO:3 and reverse primer SEQ ID NO:4, with SEQ ID NO:9 and/or SEQ ID NO:10, detected subsequently; And/or
If iii. this marker site is W2, be forward primer SEQ ID NO:5 and reverse primer SEQ ID NO:6, with SEQ ID NO:11 and/or SEQ ID NO:12, detected subsequently;
If iv. this marker site is Ni+, be forward primer SEQ ID NO:13 and reverse primer SEQ ID NO:14, with SEQ ID NO:15 and/or SEQ ID NO:16, detected subsequently;
B) make step described plant and a kind of easy infection Potyvirus a) or at least one described Potyvirus represented to the pumpkin plant hybridization of medium resistance level; And
C) select an offspring from described hybridization, this offspring represents a kind of resistant phenotype to Potyvirus, and wherein said resistant phenotype separates with step described at least one marker site a).
33., according to the method for embodiment 31 or 32, this plant that wherein step is selected in a) comprises at least two and can instruct or control the genetic determinant to the resistance of Potyvirus; And this offspring who selects step c) represents a kind of Potyvirus resistant phenotype, and wherein said resistant phenotype separates with step two marker sites a).
34. according to the method for embodiment 31 or 32, this plant that wherein step is selected in a) comprises at least three can be instructed or control Potyvirus, preferably to the genetic determinant of the resistance of ZYMV and MWMV; And this offspring who selects step c), to Potyvirus, preferably ZYMV and MWMV are represented to a kind of resistant phenotype, and wherein said resistant phenotype separates with step these marker sites a).
35., according to the method for any previous embodiment 31 to 34, wherein step this donor plant a) is a kind of plant of any previous embodiment 1 to 13.
36., according to the method for embodiment 31 to 35, comprise and make step c) in this virus resistance plant and the step b that obtain) this susceptible pumpkin plant or the additional step that backcrosses of medium resistance pumpkin plant.
37. the method for generation of the hybrid seed of the pumpkin that Potyvirus is had to resistance, comprise
A) plant a male sterile female and one and malely can educate plant, wherein said male sterile female or male to educate one of plant be a kind of plant according to any previous embodiment 1 to 13,
B) realize cross pollination between two strains,
C) make this plant growth until setting,
D) collect these fruits; And
E) obtain these hybrid seeds.
38. one kind for obtaining the method for Potyvirus resistance pumpkin plant, comprises
A) method by any previous embodiment obtain any previous embodiment plant or;
B) make plant or the medium resistance pumpkin plant hybridization of described plant and a kind of easy infection Potyvirus;
C) save by step b) an embryo producing of this hybridization;
D) from step c) described embryo bear again a kind of plant; And
E) select Potyvirus, preferably to the steps d that one of at least there is resistance in ZYMV and MWMV) a Plants.
39. one kind has the method for the pumpkin fruit of resistance for obtaining to Potyvirus, comprises
A) sow a kind of seed according to any one or plant that obtain by any one method according in any previous embodiment in any previous embodiment; And
B) make described plant growth in order to produce fruit, and results are by this fruit of described plant generation.
40. one kind can be instructed or control the genetic determinant to the resistance of Potyvirus, wherein said genetic determinant is obtainable from the genome of field pumpkin cultivation kind 268NiW, wherein
A) existing corresponding genetic determinant complementation in this genetic determinant and field pumpkin cultivation kind 268NiW, the representative seed of this field pumpkin cultivation kind 268NiW is kept at NCIMB with accession number NCIMB41727, described corresponding genetic determinant and marker site ZN genetic linkage, this marker site and Potyvirus resistance trait, preferably with a kind of ZYMV and PRSV resistance trait be divided into from, and can be by with following Oligonucleolide primers, amplification of DNA fragments being identified it in PCR: forward primer SEQ ID NO:1 and reverse primer SEQ ID NO:2, with SEQ ID NO:7 and/or SEQ ID NO:8, detected subsequently, and/or
B) at least one complementation in existing corresponding genetic determinant in this genetic determinant and field pumpkin cultivation kind 268NiW, the representative seed of this field pumpkin cultivation kind 268NiW is kept at NCIMB with accession number NCIMB41727, described corresponding genetic determinant and marker site W1 and/or W2 genetic linkage, this marker site and Potyvirus resistance trait, preferably with a kind of MWMV resistance trait be divided into from, and can be by with following Oligonucleolide primers, amplification of DNA fragments being identified it in PCR: forward primer SEQ ID NO:3 and reverse primer SEQ ID NO:4, with SEQ ID NO:9 and/or SEQ ID NO:10, detected subsequently,
And/or forward primer SEQ ID NO:5 and reverse primer SEQ ID NO:6, with SEQ ID NO:11 and/or SEQ ID NO:12, detected subsequently; And/or
C) existing corresponding genetic determinant complementation in this genetic determinant and field pumpkin cultivation kind 268NiW, the representative seed of this field pumpkin cultivation kind 268NiW is kept at NCIMB with accession number NCIMB41727, described corresponding genetic determinant and marker site Ni+ genetic linkage, this marker site and Potyvirus resistance trait, preferably with a kind of ZYMV resistance trait, more preferably with a kind of ZYMV Strain Nivir resistance trait be divided into from, and can be by with following Oligonucleolide primers, amplification of DNA fragments being identified it in PCR: forward primer SEQ ID NO:13 and reverse primer SEQ ID NO:14, with SEQ ID NO:15 and/or SEQ ID NO:16, detected subsequently.
41. an evaluation comprises at least one and can instruct or control Potyvirus, preferably to the method for the pumpkin plant of the genetic determinant of the resistance one of at least in ZYMV and MWMV, wherein said genetic determinant be obtainable from the genome of field pumpkin cultivation kind 268NiW and with at least one marker site genetic linkage, at least one in this at least one marker site and described Potyvirus resistant phenotype be divided into from, and can be in PCR with comprising that at least one following PCR Oligonucleolide primers is to by its evaluation:
If i. this marker site is ZN, be forward primer SEQ ID NO:1 and reverse primer SEQ ID NO:2, with SEQ ID NO:7 and/or SEQ ID NO:8, detected subsequently; Or
If ii. this marker site is W1, be forward primer SEQ ID NO:3 and reverse primer SEQ ID NO:4, with SEQ ID NO:9 and/or SEQ ID NO:10, detected subsequently; Or
If iii. this marker site is W2, be forward primer SEQ ID NO:5 and reverse primer SEQ ID NO:6, with SEQ ID NO:11 and/or SEQ ID NO:12, detected subsequently; Or
If iv. this marker site is Ni+, be forward primer SEQ ID NO:13 and reverse primer SEQ ID NO:14, with SEQ ID NO:15 and/or SEQ ID NO:16, detected subsequently.
42. derive from the purposes according to the Potyvirus resistance propagating materials of the pumpkin plant of any one previous embodiment, for growing into a kind of Potyvirus resistance pumpkin plant in order to produce fruit and gather in the crops described fruit.
Example
Example 1
In pumpkin with the discovery of ZYMV and the closely linked mark of MWMV resistant gene
1.1 material
The purpose of finding for mark, produce and a plurality ofly can isolate ZYMV(R strain [PP452] x S strain [TOSCA] from China squash Nigeria cultivar) and MWMV(S strain [PP477] x R strain [PP477/ (PP415/ (PP419/ (Nigeria/PP432)]) F2 colony and the sampling of resistant gene; And for ZYMV and MWMV resistance, their corresponding F3 offspring is carried out to phenotype analytical accordingly.
The diversity checking group that 96 pumpkin strains and kind by the type to by corresponding to different and market segments forms carries out the predicted value that genotyping carrys out the check of assessment development.
1.2 mark is found
Colony's compartment analysis (Bulked Segregant Analysis is carried out in different F2DNA pond with contrary resistance and susceptible phenotype, BSA), this analysis is used randomly amplified polymorphic DNA (RAPD) mark (to derive from (the Operon technologies of Ao Peilong technology company of I meter Da of California, Alameda, Calif.) and (the University of British Columbia of the UBC of Vancouver, CAN, Vancouver, Canada)).In indivedual members of F2 colony, to the candidate's mark from the BSA Screening and Identification (the RAPD band shows obvious presence/absence pattern between F2R and S colony), further test chain; And select the most closely linked mark for having more specific check development.
For ZYMV, be chosen in F2 colony the single RAPD mark OPBB09_451 shown with ZYMV resistant phenotype perfect relevant (be divided into from) and be used for the development that Taqman end points reading (EPR) is checked.
For MWMV, in two different loci (QTL), the RAPD mark relevant to the MWMV resistant phenotype to be mapped, and selected optimum mark OPAR13_507 and UBC385_656, their show accordingly and QTL1 and QTL2 the highest associated (chain).
For Ni+, by these two colonies are checked order to carry out BSA again.The reference sequences of obtained reading and pumpkin is compared, and SNP detected.Then select the most closely linked SNP for being had more specific Taqman EPR check development.
1.3Taqman EPR checks development
Separate all DNA of plants according to potassium acetate+Proteinase K scheme.For the allelomorph order-checking, at 5' and the 3' of selected RAPD candidate segment, hold nearly 3 kinds of different PCR combination of primers of design.Use obtains PCR product and the DNA sequence dna of these marks from the strain of resistance and susceptible strain group.
Taqman EPR check development is based on the allele-specific SNP found in sequence group.EPR check development is to carry out according to standard guide, comprises different PCR mixtures, DNA concentration and annealing temperature are tested.Probe is FAM-MGB and VIC-MGB Taqman probe (European Genentech, Inc. (US) 460 Point San Bruno Blvd, South San Francisco, CA, 94080 (Eurogentec)).
1.4Taqman EPR verification scheme
1. extract potassium acetate+Proteinase K scheme by standard DNA and carry out the DNA isolation genome.Finally, obtain 150 μ l DNA solutions.
2. template DNA is diluted to 1/15.
3. the DNA sample that each is diluted by 4 μ l is inhaled and is moved on in the hole of independent 384PCR plate.
4. this plate is covered and centrifugal, and be placed on ice.
5. manufacture mother liquor mixture.Every secondary response is as follows:
Vegetables project mixture Platinum Volume (μ l) Ultimate density
Platinum buffer solution 10x 1 1x
MgCl 250mM 0.6 3mM
Each 2.5mM of dNTP10mM() 0.8 0.8mM(each 0.2mM)
Taq?platinum5U/μl 0.066 0.33U
ZN R allelomorph VIC-MGB-NFQ probe 10 μ M 0.1 100nM
ZN S-allele FAM-MGB-NFQ probe 10 μ M 0.1 100nM
ZN sense primer 12.5 μ M 0.16 200nM
ZN antisense primer 12.5 μ M 0.16 200nM
ROX50x 0.1 0.5x
Qsp?H 2O 2.914 ?
Cumulative volume 6 ?
6. 6 μ l mother liquor mixtures are added in each PCR plate hole (wherein having contained 4 μ l template DNAs).
7. rotation momently.
8. this 384 plate is loaded on the PCR machine.
9. the plan of the PCR based on ABI GENEAMP PCR9700-384 panel formula is as follows:
2 minutes, 94 ℃
15 seconds, 94 ℃
1 minute, 60 ℃
40X
5 minutes, 72 ℃
10. on AB17900, this plate is carried out to reading.
1.5EPR check primer and probe sequence
1.5.1.ZYMV-Nigeria
Forward primer: 5'AGGTTTCATGGGCTTTTAATGG3'(SEQ ID NO:1)
Reverse primer: 5'CGTGAGCCTAAAACGGTTAATG3'(SEQ ID NO:2)
Resistance allele specific probe: FAM-CACTTCCCAGCCCAAAT-MGB-NFQ(SEQ ID NO:7)
Susceptible allele-specific probe: VIC-CACTTTCCAGCCCAAAT-MGB-NFQ(SEQ ID NO:8)
Ni+
Forward primer: 5'TTGCATGTTCCTTGGATGGGT3'(SEQ ID NO:13)
Reverse primer: 5'GGCAACCTCTGTCCAATTTCTTTC3'(SEQ ID NO:14)
Resistance allele specific probe: FAM-AGTTGCGACTTTCCA-MGB-NFQ(SEQ ID NO:15)
Susceptible allele-specific probe: TET-AGTTGCGACTTTTCATT-MGB-NFQ(SEQ ID NO:16)
1.5.2.MWMV-Nigeria
1.5.2.1QTL1
Forward primer: 5'GGGCAAAGAAGATCTTGTCTAGAAAG3'(SEQ ID NO:3)
Reverse primer: 5'GTTTTTGTGCAGTGTGCATCTGT3'(SEQ ID NO:4)
Resistance allele specific probe: FAM-TCATTGCACCCAACATG-MGB-NFQ(SEQ ID NO:9)
Susceptible allele-specific probe: VIC-TCATTGCACTCAACATGG-MGB-NFQ(SEQ ID NO:10)
1.5.2.1QTL2
Forward primer: 5'TTGTGTTTATATGTATGTGTGCGAG3'(SEQ ID NO:5)
Reverse primer: 5'TTTCTAGATCTCAGTGTAAGAGAACACA3'(SEQ ID NO:6)
Resistance allele specific probe: FAM-TTTGTTTGCTTGAGCTGG-MGB-NFQ(SEQ ID NO:11)
Susceptible allele-specific probe: VIC-TTTGTTCGATTGAGCTGG-MGB-NFQ(SEQ ID NO:12)
Example 2
The disease testing scheme
2.1 the use of scheme
Following scheme is applicable to all viruses (CMV, ZYMV, WMV, MWMV) on pumpkin and Cucurbita and cucumber (Cucumis).
2.2 the preservation of Strain
New infected leaf (1g) is weighed.Then with scalpel by these leaves fine shred and be placed on the papery weighing tray.This weighing tray is placed on a petri diss that comprises anhydrous calcium chloride (55mm).With Parafilm, box is sealed.The weight of the title of indicator virus strain, date saved and prepared fresh leaf on box, and record the number of box.These plates are remained in the drawer " vegetables " in refrigerator.
2.3 the propagation of virus from the dehydration preparation
Sowing susceptible kind in one or more terrines.When plant during the stage, is inoculated (vide infra, the inoculation of tester) by the dehydration preparation in " cotyledon ".At 1 week, after 10 days, the first symptom will there will be, and in the invasive stage of tool of this virus.
2.4 the preparation of inoculum
Choose infected tender leaf from terrine.For 1 gram leaf, prepare 0.1 gram coal and 4cc buffer solution.Before adding this coal and last this buffer solution of interpolation, these leaves are broken into pieces.Diamond dust is spread in this mixture.Utilize the fresh leaf of 1 gram, can inoculate 2 to 3 terrines (1 terrine=80 are to 100 plants).
2.5 the inoculation of tester
These inoculums are placed on a sled.In the cotyledon stage, these plants are inoculated.With these inoculums these cotyledons that rub, per hour upgrade in case of necessity this solution.After dry 15 minutes, then give this plant watering.Reading can carry out 5 to 6 days the time after inoculation for the first time.Then can after 7 to 10 days, carry out for the second time reading to confirm and perfect information.In order to finish test, leaf is sealed in plastic sack and is placed in biological waste.The optimal temperature conditions of carrying out this test is 20 ℃ ± 2 ° of 25 ℃ in the daytime ± 2 ° and nights.
2.6 the chronicle of events
The sowing of the terrine of 0-8 days propagation
The inoculation of 0-2 days terrines
The sowing of the 0th day tester
The inoculation of 0+6 days testers
The beginning of 0+14 days readings
The destruction of 0+30 days testers
Example 3
The guide that zucchini (custard squash) is carried out to the test of Potyvirus pathology
3.1 grading guide
Following guide is used to determine ZYMV, the WMV on leaf and fruit, the degree that PRSV, MWMV infect.From the 3rd leaf stage until the maturation plant stage (thering is mellow fruit botany) from start to finish crop is carried out to reading, assessment and grading.
According to following guide, at from 1 to 9 scale, graded, the example is shown in Fig. 1 to 9.
The guide of table 1 Potyvirus test
Figure BDA0000397680000000401
Table 2. is the grading to preservation strain 268Niw based on some screenings
For ZYMV and WMV, distinguish the slight and severe form of these Strain.
Figure BDA0000397680000000402

Claims (20)

1. the cucurbita plants of a cultivation, custard squash preferably, comprising at least one can instruct or control Potyvirus, preferably to the genetic determinant of the resistance of MWMV, PRSV, WMV and/or ZYMV.
2. plant according to claim 1, wherein said genetic determinant is obtainable from the genome of China squash.
3. plant according to claim 1 and 2, comprise at least two and describedly can instruct or control Potyvirus, preferably to the genetic determinant of the resistance of ZYMV and/or MWMV.
4. plant according to claim 1 and 2, comprise at least four and describedly can instruct or control Potyvirus, preferably to the genetic determinant of the resistance of ZYMV and MWMV.
5. according to the described plant of claim 1 to 4, wherein
A. existing corresponding genetic determinant complementation in this genetic determinant and field pumpkin cultivation kind 268NiW, the representative seed of this field pumpkin cultivation kind 268NiW is kept at NCIMB with accession number NCIMB41727, described corresponding genetic determinant with marker site ZN genetic linkage, this marker site and Potyvirus resistance trait be divided into from, and can be by with following Oligonucleolide primers, amplification of DNA fragments being identified it in PCR: forward primer SEQ ID NO:1 and reverse primer SEQ ID NO:2, with SEQ ID NO:7 and/or SEQ ID NO:8, detected subsequently, and/or
B. two complementations in existing corresponding genetic determinant in this genetic determinant and field pumpkin cultivation kind 268NiW, the representative seed of this field pumpkin cultivation kind 268NiW is kept at NCIMB with accession number NCIMB41727, described corresponding genetic determinant and marker site W1 and/or W2 genetic linkage, this marker site and Potyvirus resistance trait, preferably with a kind of MWMV resistance trait be divided into from, and can be by with following Oligonucleolide primers, amplification of DNA fragments being identified it in PCR: forward primer SEQ ID NO:3 and reverse primer SEQ ID NO:4, with SEQ ID NO:9 and/or SEQ ID NO:10, detected subsequently,
And/or forward primer SEQ ID NO:5 and reverse primer SEQ ID NO:6, with SEQ ID NO:11 and/or SEQ ID NO:12, detected accordingly subsequently; And/or
C. existing corresponding genetic determinant complementation in this genetic determinant and field pumpkin cultivation kind 268NiW, the representative seed of this field pumpkin cultivation kind 268NiW is kept at NCIMB with accession number NCIMB41727, described corresponding genetic determinant and marker site Ni+ genetic linkage, this marker site and Potyvirus resistance trait, preferably with a kind of ZYMV resistance trait be divided into from, and can be by with following Oligonucleolide primers, amplification of DNA fragments being identified it in PCR: forward primer SEQ ID NO:13 and reverse primer SEQ ID NO:14, with SEQ ID NO:15 and/or SEQ ID NO:16, detected subsequently.
6. according to the described plant of claim 1 to 5, wherein
A. existing corresponding genetic determinant complementation in this genetic determinant and field pumpkin cultivation kind 268NiW, the representative seed of this field pumpkin cultivation kind 268NiW is kept at NCIMB with accession number NCIMB41727, described corresponding genetic determinant and marker site ZN genetic linkage, this marker site and Potyvirus resistance trait be divided into from, and can be in PCR by with following Oligonucleolide primers to amplification of DNA fragments in field pumpkin cultivation kind 268NiW genome by its evaluation: forward primer SEQ ID NO:1 and reverse primer SEQ ID NO:2, with SEQ ID NO:7 and/or SEQ ID NO:8, detected subsequently, and/or
B. two complementations in existing corresponding genetic determinant in this genetic determinant and field pumpkin cultivation kind 268NiW, the representative seed of this field pumpkin cultivation kind 268NiW is kept at NCIMB with accession number NCIMB41727, described corresponding genetic determinant and marker site W1 and/or W2 genetic linkage, this marker site and Potyvirus resistance trait, preferably with a kind of MWMV resistance trait be divided into from, and can be in PCR by with following Oligonucleolide primers to amplification of DNA fragments in field pumpkin cultivation kind 268NiW genome by its evaluation: forward primer SEQ ID NO:3 and reverse primer SEQ ID NO:4, with SEQ ID NO:9 and/or SEQ ID NO:10, detected subsequently,
And/or forward primer SEQ ID NO:5 and reverse primer SEQ ID NO:6, with SEQ ID NO:11 and/or SEQ ID NO:12, detected accordingly subsequently; And/or
C. existing corresponding genetic determinant complementation in this genetic determinant and field pumpkin cultivation kind 268NiW, the representative seed of this field pumpkin cultivation kind 268NiW is kept at NCIMB with accession number NCIMB41727, described corresponding genetic determinant and marker site ZN genetic linkage, this marker site and Potyvirus resistance trait, preferably with a kind of ZYMV resistance trait be divided into from, and can be in PCR by with following Oligonucleolide primers to amplification of DNA fragments in field pumpkin cultivation kind 268NiW genome by its evaluation: forward primer SEQ ID NO:13 and reverse primer SEQ ID NO:14, with SEQ ID NO:15 and/or SEQ ID NO:16, detected subsequently.
7. according to the described plant of claim 1 to 5, wherein
A. this genetic determinant and marker site ZN genetic linkage, this marker site and Potyvirus resistance trait, preferably with a kind of ZYMV resistance trait be divided into from, and can be in PCR by with following Oligonucleolide primers to amplification of DNA fragments in described Plant Genome by its evaluation: forward primer SEQ ID NO:1 and reverse primer SEQ ID NO:2, with SEQ ID NO:7 and/or SEQ ID NO:8, detected subsequently; And/or
B. this genetic determinant and marker site W1 and/or W2 genetic linkage, this marker site and Potyvirus resistance trait, preferably with a kind of MWMV resistance trait be divided into from, and can be in PCR by with following Oligonucleolide primers to amplification of DNA fragments in described Plant Genome by its evaluation: forward primer SEQ ID NO:3 and reverse primer SEQ ID NO:4, with SEQ ID NO:9 and/or SEQ ID NO:10, detected subsequently;
And/or forward primer SEQ ID NO:5 and reverse primer SEQ ID NO:6, with SEQ ID NO:11 and/or SEQ ID NO:12, detected accordingly subsequently;
C. this genetic determinant and marker site Ni+ genetic linkage, this marker site and Potyvirus resistance trait, preferably with a kind of ZYMV resistance trait be divided into from, and can be in PCR by with following Oligonucleolide primers to amplification of DNA fragments in described Plant Genome by its evaluation: forward primer SEQ ID NO:13 and reverse primer SEQ ID NO:14, with SEQ ID NO:15 and/or SEQ ID NO:16, detected subsequently.
8. according to the described plant of claim 1 to 7, wherein should (these) genetic determinant be recessive inheritance and separate independently of one another.
9. according to the described plant of claim 1 to 8, wherein this plant is the pumpkin plant.
10. the seed of pumpkin plant according to claim 9, this seed can grow into a kind of Potyvirus resistance pumpkin plant.
11. the purposes of seed according to claim 10, for growing into a kind of Potyvirus resistance pumpkin plant.
12. select Potyvirus, preferably, to the method that one of at least represents the pumpkin plant of resistance in ZYMV and MWMV, comprise the following steps:
A. identify that comprising at least one can instruct or control the pumpkin plant to the genetic determinant of the resistance of Potyvirus, wherein said genetic determinant be obtainable from the genome of field pumpkin cultivation kind 268NiW and with at least one marker site genetic linkage, at least one in this at least one marker site and described Potyvirus resistant phenotype be divided into from, and can be in PCR with comprising that at least one following PCR Oligonucleolide primers is to by its evaluation:
I. forward primer SEQ ID NO:1 and reverse primer SEQ ID NO:2, detected with SEQ ID NO:7 and/or SEQ ID NO:8 subsequently; And/or
Ii. forward primer SEQ ID NO:3 and reverse primer SEQ ID NO:4, detected with SEQ ID NO:9 and/or SEQ ID NO:10 subsequently; And/or
Iii. forward primer SEQ ID NO:5 and reverse primer SEQ ID NO:6, detected with SEQ ID NO:11 and/or SEQ ID NO:12 subsequently; And/or
Iv. forward primer SEQ ID NO:13 and reverse primer SEQ ID NO:14, detected with SEQ ID NO:15 and/or SEQ ID NO:16 subsequently; And/or
V. a forward and reverse primer, can identify with resistance to Potyvirus be divided into from marker site.
13. according to claim 12 for selecting Potyvirus, preferably, to the method that one of at least represents the pumpkin plant of resistance in ZYMV and MWMV, comprise the following steps:
A. identify that comprising at least one can instruct or control Potyvirus, preferably to the pumpkin plant of the genetic determinant of the resistance one of at least in ZYMV and MWMV, wherein said genetic determinant be obtainable from the genome of field pumpkin cultivation kind 268NiW and with at least one marker site genetic linkage, at least one in this at least one marker site and described Potyvirus resistant phenotype be divided into from, and can be in PCR with comprising that at least one following PCR Oligonucleolide primers is to by its evaluation:
If i. this marker site is ZN, be forward primer SEQ ID NO:1 and reverse primer SEQ ID NO:2, with SEQ ID NO:7 and/or SEQ ID NO:8, detected subsequently; And/or
If ii. this marker site is W1, be forward primer SEQ ID NO:3 and reverse primer SEQ ID NO:4, with SEQ ID NO:9 and/or SEQ ID NO:10, detected subsequently; And/or
If iii. this marker site is W2, be forward primer SEQ ID NO:5 and reverse primer SEQ ID NO:6, with SEQ ID NO:11 and/or SEQ ID NO:12, detected subsequently;
If iv. this marker site is Ni+, be forward primer SEQ ID NO:13 and reverse primer SEQ ID NO:14, with SEQ ID NO:15 and/or SEQ ID NO:16, detected subsequently.
14. method according to claim 13, the plant of wherein selecting in a) in step comprises at least three can be instructed or control Potyvirus, preferably to the genetic determinant of the resistance of ZYMV and MWMV; And at step c) in the offspring that selects to Potyvirus, preferably ZYMV and MWMV are represented to a kind of resistant phenotype, and wherein said resistant phenotype separates with step three marker sites a).
15., according to the described method of claim 13 to 14, the pumpkin plant of wherein identifying in a) in step is a kind of according to the described plant of claim 1 to 9.
16., according to claim 14 and 15 described methods, comprise and making at step c) in the virus resistance plant that obtains with at step b) susceptible pumpkin plant or the additional step that backcrosses of medium resistance pumpkin plant.
17. the method for generation of the hybrid seed of the pumpkin that Potyvirus is had to resistance, comprise
A. plant and plant the male sterile female and malely can educate plant, wherein said male sterile female or male to educate one of plant be a kind of according to the described plant of any one in claim 1 to 9,
B. realize cross pollination between two strains,
C. make this plant growth until setting,
D. collect these fruits; And
E. obtain these hybrid seeds.
18. one kind for obtaining the method for Potyvirus resistance pumpkin plant, comprises
A., nonactive embryo between kind is provided, and this embryo obtains by making the described plant of claim 1 to 10 and another plant hybridization;
B. save described embryo;
C. from step c) described embryo bear again a kind of plant; And
D. select a kind of to Potyvirus, preferably to the steps d that one of at least there is resistance in ZYMV, MWMV) plant.
19. one kind can be instructed or control the genetic determinant to the resistance of Potyvirus, wherein said genetic determinant is obtainable from the genome of field pumpkin cultivation kind 268NiW, wherein
A. existing corresponding genetic determinant complementation in this genetic determinant and field pumpkin cultivation kind 268NiW, the representative seed of this field pumpkin cultivation kind 268NiW is kept at NCIMB with accession number NCIMB41727, described corresponding genetic determinant and marker site ZN genetic linkage, this marker site and Potyvirus resistance trait, preferably with a kind of ZYMV resistance trait be divided into from, and can be by with following Oligonucleolide primers, amplification of DNA fragments being identified it in PCR: forward primer SEQ ID NO:1 and reverse primer SEQ ID NO:2, with SEQ ID NO:7 and/or SEQ ID NO:8, detected subsequently, and/or
B. at least one complementation in existing corresponding genetic determinant in this genetic determinant and field pumpkin cultivation kind 268NiW, the representative seed of this field pumpkin cultivation kind 268NiW is kept at NCIMB with accession number NCIMB41727, described corresponding genetic determinant and marker site W2 genetic linkage, this marker site and Potyvirus resistance trait, preferably with a kind of MWMV resistance trait be divided into from, and can be by with following Oligonucleolide primers, amplification of DNA fragments being identified it in PCR: forward primer SEQ ID NO:5 and reverse primer SEQ ID NO:6, with SEQ ID NO:11 and/or SEQ ID NO:12, detected subsequently, and/or
C. existing corresponding genetic determinant complementation in this genetic determinant and field pumpkin cultivation kind 268NiW, the representative seed of this field pumpkin cultivation kind 268NiW is kept at NCIMB with accession number NCIMB41727, described corresponding genetic determinant and marker site Ni+ genetic linkage, this marker site and Potyvirus resistance trait, preferably with a kind of ZYMV resistance trait be divided into from, and can be by with following Oligonucleolide primers, amplification of DNA fragments being identified it in PCR: forward primer SEQ ID NO:13 and reverse primer SEQ ID NO:14, with SEQ ID NO:15 and/or SEQ ID NO:16, detected subsequently.
20. an evaluation comprises at least one and can instruct or control Potyvirus, preferably to the method for the pumpkin plant of the genetic determinant of the resistance one of at least in ZYMV and MWMV, wherein said genetic determinant be obtainable from the genome of field pumpkin cultivation kind 268NiW and with at least one marker site genetic linkage, at least one in this at least one marker site and described Potyvirus resistant phenotype be divided into from, and can be in PCR with comprising that at least one following PCR Oligonucleolide primers is to by its evaluation:
If i. this marker site is ZN, be forward primer SEQ ID NO:1 and reverse primer SEQ ID NO:2, with SEQ ID NO:7 and/or SEQ ID NO:8, detected subsequently; Or
If ii. this marker site is W1, be forward primer SEQ ID NO:3 and reverse primer SEQ ID NO:4, with SEQ ID NO:9 and/or SEQ ID NO:10, detected subsequently; And/or
If iii. this marker site is W2, be forward primer SEQ ID NO:5 and reverse primer SEQ ID NO:6, with SEQ ID NO:11 and/or SEQ ID NO:12, detected subsequently; And/or
If iv. this marker site is Ni+, be forward primer SEQ ID NO:13 and reverse primer SEQ ID NO:14, with SEQ ID NO:15 and/or SEQ ID NO:16, detected subsequently.
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